Let-7 and miR-103/107 were found to be strongly induced microRNAs (HRMs) in vascular endothelial cells under hypoxic conditions. Bioinformatics analysis predicted that these HRMs target the 3' UTR of argonaute 1 (AGO1) mRNA. Experimental validation confirmed that the HRMs are induced by HIF1α and directly bind to the AGO1 3' UTR, reducing AGO1 protein levels. This translational de-repression of AGO1 increased VEGF expression and promoted angiogenesis both in vitro and in vivo. The findings suggest that HRMs play a role in hypoxia-induced gene expression and angiogenesis by targeting AGO1.
Cre recombinase expression in cardiomyocytes using the αMHC-MerCreMer mouse model induces dose-dependent cardiac toxicity and lethality when higher doses of tamoxifen are used to increase recombination efficiency. Moderate and high tamoxifen doses caused heart failure and death in the mice through a DNA damage response leading to cardiomyocyte apoptosis and fibrosis. Lower tamoxifen doses achieved near maximal 80% recombination rates without toxicity. The optimal dose for recombination while minimizing effects was determined to be 30 μg tamoxifen/g body weight injected over three days. The study provides insights into the cellular mechanisms of cardiac Cre toxicity and an improved protocol for the αMHC-MerCreMer system.
Molecular mechanism of monoamine oxidase A gene regulation under inflammation...DR. VINAYAK GUPTA
This study identified key transcription factors that regulate the expression of monoamine oxidase A (MAOA) gene. Through deletion and reporter assays, the authors identified a core promoter region from -71 to -40 bp that is essential for basal MAOA transcription. Computational analysis predicted binding sites in this region for the transcription factors GATA2, Sp1, and TBP. Experiments showed that overexpression of these factors augmented MAOA promoter activity, while knockdown reduced endogenous and transfected MAOA expression. Additionally, the study provided evidence that Sp1 mediates MAOA expression changes under pathological conditions like inflammation and ischemia.
Understanding the effects of steroid hormone exposure on direct gene regulati...Neil Raden
The document discusses the effects of steroid hormone exposure on direct gene regulation through three main mechanisms: DNA methylation, histone methylation and acetylation, and RNA interference. It proposes that changes in physiologic reproductive hormone templates of exposure and timing, such as seen in the normal female menstrual cycle, can affect fertility and cancer risk by silencing or amplifying gene products. Specifically, it hypothesizes that mimicking the normal fluctuating estradiol and progesterone cycles through exogenous hormone replacement therapy may help restore normal gene expression and lower risks of chronic illnesses.
This document discusses the potential immunomodulatory effects of statins in organ transplant recipients. It notes that while immunosuppressive drugs prevent acute rejection, they can cause hyperlipidemia which may promote chronic rejection. Studies have found statins improve outcomes when given to transplant recipients by lowering lipids. The document then explores the biological mechanisms of statins including inhibiting HMG-CoA reductase and isoprenoid production, which can suppress T-cell response and lower MHC expression on antigen presenting cells like endothelial cells. This may prevent rejection by inhibiting interactions between MHC, T-cell receptors, and adhesion molecules like LFA-1 and ICAM-1. Further research is still needed to better understand how cholesterol synthesis
Mesenchymal stem cells (MSCs) show promise for treating immune disorders and degenerative diseases. However, targeting MSCs to damaged tissue sites is challenging. The researchers hypothesized that engineering MSCs to express leukocyte adhesion molecules could enhance their homing ability. They coupled a recombinant form of P-selectin glycoprotein ligand-1 (PSGL-1), which mediates leukocyte rolling, to MSCs. This non-covalently coupled the PSGL-1 to the MSC surface. MSCs modified with PSGL-1 were then able to tether and roll on endothelial cells under flow, mimicking leukocyte behavior, suggesting this approach may help target MSCs to sites of injury and inflammation.
This study investigated genetic polymorphisms related to homocysteine metabolism in a Pakistani population and their relationship to homocysteine levels and hyperhomocysteinemia risk. The study genotyped 6 polymorphisms in 872 subjects, finding the MTHFR C677T polymorphism was associated with higher homocysteine levels, while the MS A2756G and CBS 844ins68 polymorphisms were associated with lower levels. Individuals with the MTHFR 677TT genotype had 10 times higher odds of hyperhomocysteinemia compared to the 677CC genotype. Risk of hyperhomocysteinemia increased for those with the MTHFR 677CT or TT genotypes and folate/
Lung cancer 2011 talk-for 17-10-2011-17-10-2011Cheng Luo
This document discusses metagenes obtained from genome-wide association studies (GWAS) of lung cancer and their potential use as prognostic and personalized treatment biomarkers. Specifically, it mentions:
1. MAGE family genes obtained from GWAS can serve as immunotherapy targets for lung cancer. Previous studies have found MAGE expression correlates with survival in lung cancer patients.
2. Ongoing clinical trials are investigating MAGE-A3 vaccines as adjuvant therapy for resected early-stage lung cancers that express MAGE-A3, with the goal of improving disease-free and overall survival. Phase II trials found trends towards improved outcomes with MAGE-A3 vaccination.
3. MAGE expression in lung cancers
This study identified a recurrent amplification of the VEGFA gene locus in a subset of mouse and human hepatocellular carcinomas (HCC). Tumors with this amplification (Amppos) displayed distinct characteristics including higher proliferation, vessel density, and macrophage content compared to tumors without the amplification (Ampneg). Additionally, Amppos tumors had increased expression of VEGFA and other genes in the amplified region. Treatment with sorafenib, which targets VEGFR, was found to be particularly effective in inhibiting the growth of Amppos HCCs both in mouse models and human patients. This suggests VEGFA amplification could serve as a biomarker to identify HCC patients most likely to benefit from sorafenib or other
Cre recombinase expression in cardiomyocytes using the αMHC-MerCreMer mouse model induces dose-dependent cardiac toxicity and lethality when higher doses of tamoxifen are used to increase recombination efficiency. Moderate and high tamoxifen doses caused heart failure and death in the mice through a DNA damage response leading to cardiomyocyte apoptosis and fibrosis. Lower tamoxifen doses achieved near maximal 80% recombination rates without toxicity. The optimal dose for recombination while minimizing effects was determined to be 30 μg tamoxifen/g body weight injected over three days. The study provides insights into the cellular mechanisms of cardiac Cre toxicity and an improved protocol for the αMHC-MerCreMer system.
Molecular mechanism of monoamine oxidase A gene regulation under inflammation...DR. VINAYAK GUPTA
This study identified key transcription factors that regulate the expression of monoamine oxidase A (MAOA) gene. Through deletion and reporter assays, the authors identified a core promoter region from -71 to -40 bp that is essential for basal MAOA transcription. Computational analysis predicted binding sites in this region for the transcription factors GATA2, Sp1, and TBP. Experiments showed that overexpression of these factors augmented MAOA promoter activity, while knockdown reduced endogenous and transfected MAOA expression. Additionally, the study provided evidence that Sp1 mediates MAOA expression changes under pathological conditions like inflammation and ischemia.
Understanding the effects of steroid hormone exposure on direct gene regulati...Neil Raden
The document discusses the effects of steroid hormone exposure on direct gene regulation through three main mechanisms: DNA methylation, histone methylation and acetylation, and RNA interference. It proposes that changes in physiologic reproductive hormone templates of exposure and timing, such as seen in the normal female menstrual cycle, can affect fertility and cancer risk by silencing or amplifying gene products. Specifically, it hypothesizes that mimicking the normal fluctuating estradiol and progesterone cycles through exogenous hormone replacement therapy may help restore normal gene expression and lower risks of chronic illnesses.
This document discusses the potential immunomodulatory effects of statins in organ transplant recipients. It notes that while immunosuppressive drugs prevent acute rejection, they can cause hyperlipidemia which may promote chronic rejection. Studies have found statins improve outcomes when given to transplant recipients by lowering lipids. The document then explores the biological mechanisms of statins including inhibiting HMG-CoA reductase and isoprenoid production, which can suppress T-cell response and lower MHC expression on antigen presenting cells like endothelial cells. This may prevent rejection by inhibiting interactions between MHC, T-cell receptors, and adhesion molecules like LFA-1 and ICAM-1. Further research is still needed to better understand how cholesterol synthesis
Mesenchymal stem cells (MSCs) show promise for treating immune disorders and degenerative diseases. However, targeting MSCs to damaged tissue sites is challenging. The researchers hypothesized that engineering MSCs to express leukocyte adhesion molecules could enhance their homing ability. They coupled a recombinant form of P-selectin glycoprotein ligand-1 (PSGL-1), which mediates leukocyte rolling, to MSCs. This non-covalently coupled the PSGL-1 to the MSC surface. MSCs modified with PSGL-1 were then able to tether and roll on endothelial cells under flow, mimicking leukocyte behavior, suggesting this approach may help target MSCs to sites of injury and inflammation.
This study investigated genetic polymorphisms related to homocysteine metabolism in a Pakistani population and their relationship to homocysteine levels and hyperhomocysteinemia risk. The study genotyped 6 polymorphisms in 872 subjects, finding the MTHFR C677T polymorphism was associated with higher homocysteine levels, while the MS A2756G and CBS 844ins68 polymorphisms were associated with lower levels. Individuals with the MTHFR 677TT genotype had 10 times higher odds of hyperhomocysteinemia compared to the 677CC genotype. Risk of hyperhomocysteinemia increased for those with the MTHFR 677CT or TT genotypes and folate/
Lung cancer 2011 talk-for 17-10-2011-17-10-2011Cheng Luo
This document discusses metagenes obtained from genome-wide association studies (GWAS) of lung cancer and their potential use as prognostic and personalized treatment biomarkers. Specifically, it mentions:
1. MAGE family genes obtained from GWAS can serve as immunotherapy targets for lung cancer. Previous studies have found MAGE expression correlates with survival in lung cancer patients.
2. Ongoing clinical trials are investigating MAGE-A3 vaccines as adjuvant therapy for resected early-stage lung cancers that express MAGE-A3, with the goal of improving disease-free and overall survival. Phase II trials found trends towards improved outcomes with MAGE-A3 vaccination.
3. MAGE expression in lung cancers
This study identified a recurrent amplification of the VEGFA gene locus in a subset of mouse and human hepatocellular carcinomas (HCC). Tumors with this amplification (Amppos) displayed distinct characteristics including higher proliferation, vessel density, and macrophage content compared to tumors without the amplification (Ampneg). Additionally, Amppos tumors had increased expression of VEGFA and other genes in the amplified region. Treatment with sorafenib, which targets VEGFR, was found to be particularly effective in inhibiting the growth of Amppos HCCs both in mouse models and human patients. This suggests VEGFA amplification could serve as a biomarker to identify HCC patients most likely to benefit from sorafenib or other
1) Overexpression of microRNA-146a (miR-146a) in myeloid cells decreases osteoclast differentiation and bone erosion in rheumatoid arthritis (RA) by targeting TRAF6 and IRAK1 proteins critical for osteoclastogenesis.
2) The researcher aims to evaluate the effects of overexpressing miR-146a specifically in myeloid cells in a mouse model of RA induced by mBSA and IL-1β injections using a genetic mouse model that allows tamoxifen-inducible overexpression of miR-146a in myeloid cells.
3) The researcher hypothesizes that overexpressing miR-146a in myeloid cells prior to RA induction will decrease osteocl
Sequence Characterization of Coding Regions of the Myostatin Gene (GDF8) from...Wani Ahad
The Bakerwal breed of goat in Kashmir valley is a good meat purpose breed of goat. That attains
an appreciable body weight under a low input production system. As these goats are mainly
reared by Gujjar and Bakarwal tribes of the J & K state, so they usually are fed with the weeds,
herbs, shrubs and grasses of pastures that will otherwise go waste. These goats constitute the
main livestock wealth. With the good productive and reproductive potential, it makes these animals
an important animal protein source for developing countries like India. The myostatin gene
(GDF8) is important in the physiology of stock animals because its product produces a direct effect
on muscle development and consequently also on meat production. The myostatin sequence is
known in several mammalian species and shows a high degree of amino acid sequence conservation,
although several alterations in the intron and exon regions have been identified. The objective
of our work is to characterize the myostatin coding regions using gene sequencing and polymerase
chain reaction methods of Capra hircus (Bakerwal breed) and to compare them with the
Ovis aries and other livestock species of animal, looking for variations in nucleotide and protein
sequences. As mutations in the myostatin gene can inactivate its expression and result in a
non-functional protein, which leads to increase in muscle growth in many species. In this way, we are able to identify 3 alterations on the presumed myostatin protein sequence as compared to non
double-muscled ovine sequences.
180425 Bioinformatic workflows to discover transposon/gene biomarkers in cancerM. Gonzalo Claros
The document describes a workflow called NearTrans that identifies differentially expressed transposable elements (TEs) in prostate cancer. NearTrans analyzes RNA-seq data from normal and tumor samples to detect 5 TEs, including L1PA3, L1PA4, and L1PA7, that are upregulated in prostate cancer. It also examines the closest gene to each differentially expressed TE and finds some correlations between TE and gene expression, such as for HERV17-int and the nearby ACSM1 gene.
ABO Gene Polymorphism and Thrombomodulin −33G>A Polymorphism Were Not Risk Fa...UniversitasGadjahMada
This research article studied the relationship between ABO gene polymorphism, thrombomodulin gene polymorphism, and the risk of acute myocardial infarction (AMI) in Javanese men in Indonesia. The study analyzed 192 subjects and found that neither ABO polymorphism nor thrombomodulin polymorphism -33G>A were risk factors for AMI. Previous studies on other populations had found associations, but this study on Javanese men did not replicate those findings, possibly due to ethnic differences in genetics. The researchers concluded that in this population, the two polymorphisms studied did not increase the risk of AMI.
This study investigated the effects of cartilage oligomeric matrix protein (COMP) on gene and protein expression in human vascular smooth muscle cells. The researchers found that exposing the cells to COMP significantly upregulated the expression of four genes implicated in peripheral arterial disease: ANGPTL4, IL-8, IL-12A, and THBS2. The purpose of the study was to determine the effects of COMP on the expression of Group B thrombospondin genes and proteins as well as several other proteins. The researchers hypothesized that COMP would upregulate specific genes and proteins. They used qrt-PCR and quantitative immunoassays to analyze gene and protein expression changes in response to COMP exposure.
Proteomic profiling of fractionated post-myocardial infarctionExpedeon
Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide.Heart failure is the result of adverse remodeling of the collagenous scar that replaces the
damaged myocardium after MI. Markers of LV remodeling can be either identified in the circulation (e.g. serum or plasma) or detected in the heart by imaging technologies or biopsy.
- 9th American Annual Meeting of the American Society of Gene Therapy – May 2006, Baltimore, Maryland. Poster presentation: “Long Term effects of intramyocardial pcDNA3-VEGF165 transfer after experimental myocardial infarction in the rat scar tissue extracellular matrix”.
This document summarizes the bifunctional role of glycogen synthase kinase-3 beta (GSK-3β) in regulating cell death pathways. GSK-3β can both promote and inhibit apoptosis depending on cell type and signaling environment. It promotes apoptosis by inhibiting pro-survival factors and facilitating pro-apoptotic factors like p53. However, it also inhibits apoptosis under basal conditions by promoting degradation of p53 through MDM2 and stabilizing NF-κB, which protects against tumor necrosis factor-induced cell death. Due to its complex roles in cell survival, GSK-3β is an important target for drug development in diseases like cancer.
Promoter Methylation Of Genes In And Around The 03君瑋 徐
Genetic, epigenetic, and computational screening approaches were used to identify 43 genes located in the 6q12-27 region for characterization of methylation status in lung cancer. Twelve genes showed methylation in lung cancer cell lines, with five genes (TCF21, SYNE1, AKAP12, IL20RA, and ACAT2) exhibiting the highest prevalence of methylation. These five genes showed similar methylation patterns in lung adenocarcinoma tumors regardless of smoking status, but their methylation was not associated with patient survival.
Expression of Glutaminase and Vesicular Glutamate Transporter Type 2 Immunore...Heith Crosby
This study examined the effects of a surgical tail incision on glutaminase (GLS) and vesicular glutamate transporter type 2 (VGluT2) expression in sacral dorsal root ganglia (DRG) neurons in rats. The researchers found that sacral DRG neurons innervate the area of surgical incision using retrograde tracing. Both GLS and VGluT2 immunoreactivity were elevated in sacral DRG neurons for up to 72 hours post-incision, while GLS mRNA levels rapidly decreased and remained depressed for at least 96 hours. The rapid depletion of GLS mRNA and subsequent elevation of GLS and VGluT2 proteins in sacral DRG neurons following surgical incision
Variant G6PD levels promote tumor cell proliferation or apoptosis via the STA...Enrique Moreno Gonzalez
Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.
This study aimed to identify domains in the N-terminal region of the human telomerase catalytic subunit (hTERT) that are required for telomerase activity and function in vivo. The researchers introduced mutations substituting six amino acid stretches throughout the N-terminal region of hTERT and assessed the ability of the mutant enzymes to maintain telomeres and support cellular proliferation. They identified four domains essential for in vitro and in vivo enzyme activity, two of which were required for binding to the hTERT RNA subunit. Additionally, they discovered a novel domain, termed DAT, where mutations left the enzyme catalytically active but unable to function in vivo, suggesting it is involved in other aspects of telomere elongation beyond catalytic
AACR Immune Infiltration In ER, PR, HER2 IHC SubtypesRafael Casiano
This study used a multiplexed immunofluorescence assay to measure levels of immune cell infiltration in different breast cancer subtypes defined by estrogen receptor, progesterone receptor, and HER2 status. The results showed that ER-/PR-/HER2 overexpressed and triple negative subtypes had the highest percentages of CD4+ and CD8+ T cells as well as Ki67+ proliferating cells. However, in the ER-/PR-/HER2 overexpressed subtype, despite high levels of CD8+ cytotoxic T cells, many tumor cells were still Ki67+. This suggests that the presence of intratumoral T cells does not necessarily indicate an active anti-tumor immune response and that immunotherapy targeting immune checkpoints may provide benefit.
This document discusses advances in multiplexed genotyping and its impact on the treatment of lung cancers. Key points include:
1) The era of "one size fits all" treatment of lung cancers is over due to advances in identifying oncogenic drivers through multiplexed testing of tumor samples.
2) Identifying these drivers through comprehensive genomic profiling allows for matching patients to targeted therapies, improving outcomes over previous untargeted approaches.
3) The MSK-IMPACT assay profiles 341 cancer genes and has identified targets for both approved and investigational targeted therapies, improving personalized treatment of lung cancer patients.
The DIONESUS algorithm provides a scalable and accurate method to reconstruct dynamic phosphoproteomic networks and reveal new drug targets. The document describes an experiment where the DIONESUS algorithm was applied to phosphorylation kinetics data from panels of growth factors, small molecule inhibitors, and antioxidants tested on A431 cells. Inhibition of SRC family kinases or PLCγ pathways attenuated EGF-stimulated cell death, while inhibition of PI3K, autocrine signaling pathways, or antioxidants decreased cell viability. The phosphorylation profiles informed dynamic network modeling to infer signaling pathways and potential drug targets.
This study investigated the effects of artesunate on drug resistance in lung carcinoma cell lines. The results showed that:
1) Artesunate attenuated the viability of drug-resistant lung carcinoma cell lines A549/DDP and SBC-3/ADM and suppressed the multidrug resistance protein BCRP.
2) Artesunate treatment upregulated miR-493-5p expression and downregulated the target gene BRCA1 in the drug-resistant cell lines.
3) Silencing miR-493-5p reversed the anti-drug resistance effects of artesunate by increasing cell viability and BCRP expression and upregulating the BRCA1 pathway.
Sima Lev The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBCSima Lev
1) The study identifies a signaling circuit involving AXL receptor, PYK2 kinase, and PKCα kinase (the AXL-PYK2-PKCα axis) that regulates stemness in triple-negative breast cancer (TNBC).
2) Depletion of PYK2 or PKCα reduces expression of proteins in the axis like AXL and transcription factor FRA1, and decreases formation of mammospheres and expression of cancer stem cell markers.
3) PYK2 depletion decreases AXL transcription through feedback involving FRA1 and TAZ transcription factors, while PKCα inhibition enhances AXL degradation. PYK2 and PKCα cooperate to regulate stemness pathways through the axis
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...Sima Lev
This study found that the animal lectin galectin-8 (gal-8) promotes cytokine expression and metastatic tumor growth in mice. Specifically:
- Gal-8 induces the expression and secretion of cytokines like SDF-1 and MCP-1 in various cell types through binding to a uPAR/LRP1/integrin complex that activates the JNK and NFkB pathways.
- Cytokines induced by gal-8 promote cancer cell migration toward cells treated with gal-8. Knockout mice lacking gal-8 have lower cytokine levels and reduced tumor growth and metastasis compared to normal mice.
- Gal-8 treatment of osteoblasts increases expression of cytokines that attract prostate cancer
1) The study evaluated the effects of two growth hormone receptor (GHR) mutations (p.R229H and c.899dupC) found in a patient with GH insensitivity.
2) Functional studies showed that the p.R229H mutation did not impair GHR signaling or function. However, the c.899dupC mutation resulted in a truncated GHR protein that was unable to activate downstream signaling pathways in response to GH.
3) When coexpressed, the c.899dupC mutation dominantly inhibited the normal signaling of wild-type GHR and the p.R229H variant. This dominant negative effect explains the patient's GH insensitivity phenotype.
This document summarizes a research article that studied the role of the heparan sulfate sulfotransferase 3-OST3A in breast cancer. The key findings were:
1) 3-OST3A expression was epigenetically repressed in most breast cancer cell lines through DNA methylation and histone modifications, except in HER2+ SKBR3 cells.
2) Gain and loss of function experiments showed 3-OST3A had profound effects on cell behavior, acting as a tumor suppressor in luminal MCF-7 and triple negative MDA-MB-231 cells but as an oncogene in HER2+ SKBR3 cells.
3) In a clinical study
Preterm labor is a multifactorial problem that current treatment options address only symptomatically rather than causally. Preventative treatment with progesterone can lower preterm birth rates in high-risk groups by over 30%. Tocolysis using beta-adrenergic agonists like terbutaline and fenoterol can be used to prevent premature labor by relaxing the uterus. However, these drugs often cause side effects from activating the sympathetic nervous system.
1) Overexpression of microRNA-146a (miR-146a) in myeloid cells decreases osteoclast differentiation and bone erosion in rheumatoid arthritis (RA) by targeting TRAF6 and IRAK1 proteins critical for osteoclastogenesis.
2) The researcher aims to evaluate the effects of overexpressing miR-146a specifically in myeloid cells in a mouse model of RA induced by mBSA and IL-1β injections using a genetic mouse model that allows tamoxifen-inducible overexpression of miR-146a in myeloid cells.
3) The researcher hypothesizes that overexpressing miR-146a in myeloid cells prior to RA induction will decrease osteocl
Sequence Characterization of Coding Regions of the Myostatin Gene (GDF8) from...Wani Ahad
The Bakerwal breed of goat in Kashmir valley is a good meat purpose breed of goat. That attains
an appreciable body weight under a low input production system. As these goats are mainly
reared by Gujjar and Bakarwal tribes of the J & K state, so they usually are fed with the weeds,
herbs, shrubs and grasses of pastures that will otherwise go waste. These goats constitute the
main livestock wealth. With the good productive and reproductive potential, it makes these animals
an important animal protein source for developing countries like India. The myostatin gene
(GDF8) is important in the physiology of stock animals because its product produces a direct effect
on muscle development and consequently also on meat production. The myostatin sequence is
known in several mammalian species and shows a high degree of amino acid sequence conservation,
although several alterations in the intron and exon regions have been identified. The objective
of our work is to characterize the myostatin coding regions using gene sequencing and polymerase
chain reaction methods of Capra hircus (Bakerwal breed) and to compare them with the
Ovis aries and other livestock species of animal, looking for variations in nucleotide and protein
sequences. As mutations in the myostatin gene can inactivate its expression and result in a
non-functional protein, which leads to increase in muscle growth in many species. In this way, we are able to identify 3 alterations on the presumed myostatin protein sequence as compared to non
double-muscled ovine sequences.
180425 Bioinformatic workflows to discover transposon/gene biomarkers in cancerM. Gonzalo Claros
The document describes a workflow called NearTrans that identifies differentially expressed transposable elements (TEs) in prostate cancer. NearTrans analyzes RNA-seq data from normal and tumor samples to detect 5 TEs, including L1PA3, L1PA4, and L1PA7, that are upregulated in prostate cancer. It also examines the closest gene to each differentially expressed TE and finds some correlations between TE and gene expression, such as for HERV17-int and the nearby ACSM1 gene.
ABO Gene Polymorphism and Thrombomodulin −33G>A Polymorphism Were Not Risk Fa...UniversitasGadjahMada
This research article studied the relationship between ABO gene polymorphism, thrombomodulin gene polymorphism, and the risk of acute myocardial infarction (AMI) in Javanese men in Indonesia. The study analyzed 192 subjects and found that neither ABO polymorphism nor thrombomodulin polymorphism -33G>A were risk factors for AMI. Previous studies on other populations had found associations, but this study on Javanese men did not replicate those findings, possibly due to ethnic differences in genetics. The researchers concluded that in this population, the two polymorphisms studied did not increase the risk of AMI.
This study investigated the effects of cartilage oligomeric matrix protein (COMP) on gene and protein expression in human vascular smooth muscle cells. The researchers found that exposing the cells to COMP significantly upregulated the expression of four genes implicated in peripheral arterial disease: ANGPTL4, IL-8, IL-12A, and THBS2. The purpose of the study was to determine the effects of COMP on the expression of Group B thrombospondin genes and proteins as well as several other proteins. The researchers hypothesized that COMP would upregulate specific genes and proteins. They used qrt-PCR and quantitative immunoassays to analyze gene and protein expression changes in response to COMP exposure.
Proteomic profiling of fractionated post-myocardial infarctionExpedeon
Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide.Heart failure is the result of adverse remodeling of the collagenous scar that replaces the
damaged myocardium after MI. Markers of LV remodeling can be either identified in the circulation (e.g. serum or plasma) or detected in the heart by imaging technologies or biopsy.
- 9th American Annual Meeting of the American Society of Gene Therapy – May 2006, Baltimore, Maryland. Poster presentation: “Long Term effects of intramyocardial pcDNA3-VEGF165 transfer after experimental myocardial infarction in the rat scar tissue extracellular matrix”.
This document summarizes the bifunctional role of glycogen synthase kinase-3 beta (GSK-3β) in regulating cell death pathways. GSK-3β can both promote and inhibit apoptosis depending on cell type and signaling environment. It promotes apoptosis by inhibiting pro-survival factors and facilitating pro-apoptotic factors like p53. However, it also inhibits apoptosis under basal conditions by promoting degradation of p53 through MDM2 and stabilizing NF-κB, which protects against tumor necrosis factor-induced cell death. Due to its complex roles in cell survival, GSK-3β is an important target for drug development in diseases like cancer.
Promoter Methylation Of Genes In And Around The 03君瑋 徐
Genetic, epigenetic, and computational screening approaches were used to identify 43 genes located in the 6q12-27 region for characterization of methylation status in lung cancer. Twelve genes showed methylation in lung cancer cell lines, with five genes (TCF21, SYNE1, AKAP12, IL20RA, and ACAT2) exhibiting the highest prevalence of methylation. These five genes showed similar methylation patterns in lung adenocarcinoma tumors regardless of smoking status, but their methylation was not associated with patient survival.
Expression of Glutaminase and Vesicular Glutamate Transporter Type 2 Immunore...Heith Crosby
This study examined the effects of a surgical tail incision on glutaminase (GLS) and vesicular glutamate transporter type 2 (VGluT2) expression in sacral dorsal root ganglia (DRG) neurons in rats. The researchers found that sacral DRG neurons innervate the area of surgical incision using retrograde tracing. Both GLS and VGluT2 immunoreactivity were elevated in sacral DRG neurons for up to 72 hours post-incision, while GLS mRNA levels rapidly decreased and remained depressed for at least 96 hours. The rapid depletion of GLS mRNA and subsequent elevation of GLS and VGluT2 proteins in sacral DRG neurons following surgical incision
Variant G6PD levels promote tumor cell proliferation or apoptosis via the STA...Enrique Moreno Gonzalez
Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.
This study aimed to identify domains in the N-terminal region of the human telomerase catalytic subunit (hTERT) that are required for telomerase activity and function in vivo. The researchers introduced mutations substituting six amino acid stretches throughout the N-terminal region of hTERT and assessed the ability of the mutant enzymes to maintain telomeres and support cellular proliferation. They identified four domains essential for in vitro and in vivo enzyme activity, two of which were required for binding to the hTERT RNA subunit. Additionally, they discovered a novel domain, termed DAT, where mutations left the enzyme catalytically active but unable to function in vivo, suggesting it is involved in other aspects of telomere elongation beyond catalytic
AACR Immune Infiltration In ER, PR, HER2 IHC SubtypesRafael Casiano
This study used a multiplexed immunofluorescence assay to measure levels of immune cell infiltration in different breast cancer subtypes defined by estrogen receptor, progesterone receptor, and HER2 status. The results showed that ER-/PR-/HER2 overexpressed and triple negative subtypes had the highest percentages of CD4+ and CD8+ T cells as well as Ki67+ proliferating cells. However, in the ER-/PR-/HER2 overexpressed subtype, despite high levels of CD8+ cytotoxic T cells, many tumor cells were still Ki67+. This suggests that the presence of intratumoral T cells does not necessarily indicate an active anti-tumor immune response and that immunotherapy targeting immune checkpoints may provide benefit.
This document discusses advances in multiplexed genotyping and its impact on the treatment of lung cancers. Key points include:
1) The era of "one size fits all" treatment of lung cancers is over due to advances in identifying oncogenic drivers through multiplexed testing of tumor samples.
2) Identifying these drivers through comprehensive genomic profiling allows for matching patients to targeted therapies, improving outcomes over previous untargeted approaches.
3) The MSK-IMPACT assay profiles 341 cancer genes and has identified targets for both approved and investigational targeted therapies, improving personalized treatment of lung cancer patients.
The DIONESUS algorithm provides a scalable and accurate method to reconstruct dynamic phosphoproteomic networks and reveal new drug targets. The document describes an experiment where the DIONESUS algorithm was applied to phosphorylation kinetics data from panels of growth factors, small molecule inhibitors, and antioxidants tested on A431 cells. Inhibition of SRC family kinases or PLCγ pathways attenuated EGF-stimulated cell death, while inhibition of PI3K, autocrine signaling pathways, or antioxidants decreased cell viability. The phosphorylation profiles informed dynamic network modeling to infer signaling pathways and potential drug targets.
This study investigated the effects of artesunate on drug resistance in lung carcinoma cell lines. The results showed that:
1) Artesunate attenuated the viability of drug-resistant lung carcinoma cell lines A549/DDP and SBC-3/ADM and suppressed the multidrug resistance protein BCRP.
2) Artesunate treatment upregulated miR-493-5p expression and downregulated the target gene BRCA1 in the drug-resistant cell lines.
3) Silencing miR-493-5p reversed the anti-drug resistance effects of artesunate by increasing cell viability and BCRP expression and upregulating the BRCA1 pathway.
Sima Lev The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBCSima Lev
1) The study identifies a signaling circuit involving AXL receptor, PYK2 kinase, and PKCα kinase (the AXL-PYK2-PKCα axis) that regulates stemness in triple-negative breast cancer (TNBC).
2) Depletion of PYK2 or PKCα reduces expression of proteins in the axis like AXL and transcription factor FRA1, and decreases formation of mammospheres and expression of cancer stem cell markers.
3) PYK2 depletion decreases AXL transcription through feedback involving FRA1 and TAZ transcription factors, while PKCα inhibition enhances AXL degradation. PYK2 and PKCα cooperate to regulate stemness pathways through the axis
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...Sima Lev
This study found that the animal lectin galectin-8 (gal-8) promotes cytokine expression and metastatic tumor growth in mice. Specifically:
- Gal-8 induces the expression and secretion of cytokines like SDF-1 and MCP-1 in various cell types through binding to a uPAR/LRP1/integrin complex that activates the JNK and NFkB pathways.
- Cytokines induced by gal-8 promote cancer cell migration toward cells treated with gal-8. Knockout mice lacking gal-8 have lower cytokine levels and reduced tumor growth and metastasis compared to normal mice.
- Gal-8 treatment of osteoblasts increases expression of cytokines that attract prostate cancer
1) The study evaluated the effects of two growth hormone receptor (GHR) mutations (p.R229H and c.899dupC) found in a patient with GH insensitivity.
2) Functional studies showed that the p.R229H mutation did not impair GHR signaling or function. However, the c.899dupC mutation resulted in a truncated GHR protein that was unable to activate downstream signaling pathways in response to GH.
3) When coexpressed, the c.899dupC mutation dominantly inhibited the normal signaling of wild-type GHR and the p.R229H variant. This dominant negative effect explains the patient's GH insensitivity phenotype.
This document summarizes a research article that studied the role of the heparan sulfate sulfotransferase 3-OST3A in breast cancer. The key findings were:
1) 3-OST3A expression was epigenetically repressed in most breast cancer cell lines through DNA methylation and histone modifications, except in HER2+ SKBR3 cells.
2) Gain and loss of function experiments showed 3-OST3A had profound effects on cell behavior, acting as a tumor suppressor in luminal MCF-7 and triple negative MDA-MB-231 cells but as an oncogene in HER2+ SKBR3 cells.
3) In a clinical study
Preterm labor is a multifactorial problem that current treatment options address only symptomatically rather than causally. Preventative treatment with progesterone can lower preterm birth rates in high-risk groups by over 30%. Tocolysis using beta-adrenergic agonists like terbutaline and fenoterol can be used to prevent premature labor by relaxing the uterus. However, these drugs often cause side effects from activating the sympathetic nervous system.
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
TIF1-gamma Controls Erythroid Cell Fate by Regulating Transcription ElongationJoe Lee
1) A genetic suppressor screen in zebrafish identified a mutation in the cdc73 gene that rescued the erythroid defect in tif1g (moonshine) mutants.
2) cdc73 encodes a subunit of the PAF elongation complex. Knockdown of other PAF subunits also rescued tif1g mutants, indicating TIF1g antagonizes the PAF complex.
3) Biochemical studies showed TIF1g interacts with erythroid transcription factors and elongation factors, coupling them to promote transcription elongation of erythroid genes by counteracting polymerase II pausing.
The Hedgehog (Hh) signal transduction pathway is involved in diverse physiological processes such as cell survival, proliferation, differentiation, embryonic development, and fetal determination. Perturbations in this pathway leading to its aberrant activation have been implicated in various cancers including acute myeloid leukemia (AML). AML is a highly complex hematological malignancy resulting due to accumulation of abnormal myeloblasts in the bone marrow leading to bone marrow failure and ultimately death. Recently, the role of hedgehog signaling in AML pathogenesis has been uncovered and thus pharmacological targeting of the hedgehog pathway in AML has become a prime therapeutic option. In this mini-review, we highlight the role of the hedgehog pathway in AML and review the latest drugs as well as possible Hh targets in treating AML.
This study identified cancer testes antigens (CTAs) that regulate hypoxia-inducible factor 1-alpha (HIF-1α) signaling. Researchers performed an siRNA screen of 120 CTAs using a luciferase reporter fused to a HIF response element, identifying MAGEA3/6 and IGF2BP3 as hits. Western blot analysis showed depletion of these CTAs decreased HIF-1α protein levels. This suggests CTAs may support stress signaling under hypoxic conditions and play functional roles in tumor cell survival.
1) The document examines ATF3 expression in cardiomyocytes in response to phenylephrine (PE) treatment.
2) Immunofluorescent staining of cardiomyocytes showed increased ATF3 nuclear staining after 0.5 hours of PE treatment, which decreased after 2 hours.
3) Quantitative PCR analysis found c-Jun mRNA was induced after 2 hours of PE, but ATF3, Egr1, and Fos mRNA levels remained unchanged.
The study analyzed gene expression profiles of MPN leukemia stem cells (LSCs) compared to normal hematopoietic stem cells (HSCs) to identify genes and pathways involved in MPN development. When comparing MPN LSCs to HSCs, differentially expressed genes were identified, including MAMDC2, ABCA13, IFIT2, and IL1RAP, which are involved in interferon response and cytokine signaling pathways. Analysis indicated that cytokine signaling and immune response pathways may be dysregulated in MPN LSCs. This suggests that anti-inflammatory or immunomodulatory drugs could be effective MPN treatments.
- The document examines the role of plasminogen activator inhibitor 1 (PAI-1) in the recruitment of mast cells (MCs) to glioma tumors.
- It finds that neutralizing PAI-1 attenuates the infiltration of MCs into glioma tumors. It also finds that MCs express the PAI-1 receptor LRP1, and blocking LRP1 also attenuates MC migration.
- Activation of the potential PAI-1/LRP1 axis in MCs by purified PAI-1 promotes increased phosphorylation of STAT3 and subsequent MC exocytosis. This indicates the PAI-1/LRP1 axis influences MC recruitment in glioma tumors.
This document summarizes research investigating the correlation between heparanase (HPA) expression and endothelial-to-mesenchymal transition (EndMT) in vascular remodeling. The researchers found that overexpression of HPA in endothelial cells enhanced EndMT development. In shear stress experiments, HPA overexpression increased expression of mesenchymal markers and decreased expression of endothelial markers. Treatment with cytokines that induce EndMT also increased mesenchymal markers and decreased endothelial markers in HPA overexpressing cells compared to wild type cells. Experiments using variable shear stress mimicking arterial bifurcations demonstrated a link between shear stress and EndMT in vascular remodeling. Future work will further study the relationship between the endothelial glycocalyx, shear stress, and
This document summarizes a study investigating microRNA regulatory networks in a mouse model of inherited retinal degeneration. In silico analysis predicted over 5,000 target genes for miRNAs found to be dysregulated in the mouse model. Proteomic analysis of mouse retinas identified over 1,800 proteins, with expression of over 800 proteins differing between normal and diseased retinas. 133 potential miRNA-target pairs were identified based on inverse expression patterns. The study validated interactions between miR-1 and its target Ctbp2, miR-96 and its target Slc6a9, and miR-96/182 targeting Rac1. Further analysis of the Rac1 interactome identified additional miRNAs that may target Rac1.
Advances Perspectives in Syncytin-1 From Biology to Clinical Practicessuppubs1pubs1
Syncytin-1 serves as an enveloped membrane glycoprotein encoded from env gene and expressed in placenta specifically as HERV-W member product of human genome playing an essential role in cell fusion process of from trophoblast to syncytiotrophoblast during each individual pregnancy. It is widely maintained that unusual expressive levels of syncytin-1 have close relationships to obstetrical syndromes such as pre-eclampsia as a typical gestational hypertension symptom. In this review, correlations between syncytin-1 and related diseases are in detailed discussions.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...eshaasini
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 Induced PI3K/Akt/mTor Pathway in the Regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive
cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC
directly related to the disease agressivity. Télomérase, is expressed
by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by
IL-6 is activated in the HCC. Our aim is to investigate the effect
of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/
PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...eshaasini
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Involvement of Interleukin-6 induced PI3K/Akt/mTor pathway in the regulation ...semualkaira
Hepatocellular Carcinoma (HCC) is an invasive cancer. Alphafoetoprotein (AFP) is a diagnostic marker for HCC directly related to the disease agressivity. Telomerase, is expressed by 90% of HCC. PI3K/Akt/mTOR pathway wich is regulated by IL-6 is activated in the HCC. Our aim is to investigate the effect of IL-6 on AFP and telomerase secretion in HepG2/C3A and PLC/ PRF/5 cell lines.
Here are the key biological and physiological mechanisms involved in genetically modified crops:
1. Gene insertion: The desired gene is isolated and inserted into the plant's genome using recombinant DNA technology. Common methods include Agrobacterium-mediated transformation or direct DNA delivery via particle bombardment.
2. Promoter selection: The inserted gene is regulated by selecting an appropriate promoter that controls the timing and location of gene expression. Constitutive promoters allow continuous expression in all tissues, while tissue-specific promoters restrict expression.
3. Protein expression: The inserted gene is transcribed and translated inside the plant cell, resulting in the production of a novel protein or trait. For example, Bt crops express insecticidal Cry proteins from Bacillus thuring
Annals of Mutagenesis is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Mutagenesis.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Mutagenesis. Annals of Mutagenesis accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of mutagenesis.
Annals of Mutagenesis strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
This summary analyzes the mRNA expression levels of the Gmhsp17.6-L gene in soybean genotypes that are resistant or susceptible to the root-knot nematode Meloidogyne javanica. Previous research identified a microsatellite marker, 176 Soy HSP, that strongly correlates with resistance to M. javanica. Sequencing of this marker revealed similarity to the promoter region of the Gmhsp17.6-L gene. The study examines levels of Gmhsp17.6-L mRNA transcripts in resistant and susceptible genotypes using ribonuclease protection assay and quantitative PCR. Results indicate higher mRNA levels in resistant genotypes, which had larger AT(n) insertions in the Gmhsp17
Similar to Chen et al. JCI 2013 HRM Ago1 angiogenesis (20)
1. Research article
The Journal of Clinical Investigation http://www.jci.org 1
Hypoxia-responsive miRNAs target
argonaute 1 to promote angiogenesis
Zhen Chen,1,2 Tsung-Ching Lai,3 Yi-Hua Jan,3 Feng-Mao Lin,4 Wei-Chi Wang,4 Han Xiao,1
Yun-Ting Wang,5 Wei Sun,1 Xiaopei Cui,1 Ying-Shiuan Li,1 Tzan Fang,4 Hongwei Zhao,6
Chellappan Padmanabhan,6 Ruobai Sun,1 Danny Ling Wang,5 Hailing Jin,6
Gar-Yang Chau,7 Hsien-Da Huang,4 Michael Hsiao,3 and John Y-J. Shyy1,2
1Division of Biomedical Sciences, University of California, Riverside, California, USA. 2Department of Medicine, School of Medicine, UCSD,
San Diego, California, USA. 3Genomics Research Center, Academia Sinica, Taipei, Taiwan. 4Institute of Bioinformatics and Systems Biology,
National Chiao Tung University, Hsin-Chu, Taiwan. 5Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan.
6Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA.
7Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan.
Despite a general repression of translation under hypoxia, cells selectively upregulate a set of hypoxia-inducible
genes. Results from deep sequencing revealed that Let-7 and miR-103/107 are hypoxia-responsive microRNAs
(HRMs) that are strongly induced in vascular endothelial cells. In silico bioinformatics and in vitro validation
showed that these HRMs are induced by HIF1α and target argonaute 1 (AGO1), which anchors the microRNA-
induced silencing complex (miRISC). HRM targeting of AGO1 resulted in the translational desuppression of
VEGF mRNA. Inhibition of HRM or overexpression of AGO1 without the 3′ untranslated region decreased
hypoxia-induced angiogenesis. Conversely, AGO1 knockdown increased angiogenesis under normoxia in vivo.
In addition, data from tumor xenografts and human cancer specimens indicate that AGO1-mediated transla-
tional desuppression of VEGF may be associated with tumor angiogenesis and poor prognosis. These findings
provide evidence for an angiogenic pathway involving HRMs that target AGO1 and suggest that this pathway
may be a suitable target for anti- or proangiogenesis strategies.
Introduction
Hypoxic stress induces various physiological or pathophysiologi-
cal responses in conditions including high-altitude adaptations,
development, wound healing, ischemic heart disease, advanced
atherosclerosis, stroke, and tumorigenesis. These multifaceted
changes are controlled by a profound but coordinated gene expres-
sion profile, signified by a panel of hypoxia-inducible genes, despite
a nominal inhibition of general protein synthesis. Among these
genes, hypoxia-inducible factor 1α (HIF1α) is a key transcription
factor (TF) robustly induced by oxygen deprivation to increase
hundreds of proteins involved in angiogenesis, erythropoiesis, cell-
cycle arrest, and metastasis (1, 2). Consensus hypoxia-responsive
elements (HREs) in the promoter region are responsible for the
HIF1α transcriptional induction of approximately 70 genes (3).
However, mechanisms at the posttranscriptional level involving the
3′ UTR of mRNAs are also important for the hypoxia induction of
genes, especially for genes lacking HREs. A synergism between tran-
scriptional induction and posttranscriptional mechanisms seems
crucial for the optimal induction of hypoxia-inducible genes (1, 4).
For example, hypoxia can upregulate VEGF via HIF1α-dependent
and -independent transcription (5, 6), 5′-cap–independent, internal
ribosomal entry site–mediated (IRES-mediated) preferential trans-
lation (7), and increased mRNA stability (8, 9).
MicroRNAs (miRNAs) are noncoding small RNAs that regulate
gene expression at the posttranscriptional level. After being pro-
cessed from their hairpin precursors, miRNAs are loaded into the
miRNA-induced silencing complex (miRISC). Consequently, Argo-
naute (AGO, also known as eukaryotic initiation factor 2C, eIF2C),
the core protein in miRISC, tethers miRNA to the 3′ UTR of the tar-
get mRNAs, which results in mRNA degradation or translational
repression (10, 11). Among AGO protein family members expressed
in human cells, AGO1–AGO4 are involved in small RNA-induced
gene silencing, and only AGO1 and AGO2 exhibit strand-dissoci-
ating activity of miRNA duplexes (12–14). Despite ample studies
focusing on miRNAs responding to various stress conditions, little
is known about the regulation of AGO expression under stress con-
ditions and its functional consequences in the vasculature.
Several miRNAs are regulated by hypoxia and thus termed
hypoxia-responsive miRNA (HRM) or hypoxamiRs (15–18). A sub-
set of HRMs is considered proangiogenic (19). However, the molec-
ular mechanisms by which HRMs promote angiogenesis remain
elusive. Using sequencing-by-synthesis (SBS) deep sequencing, we
generated a comprehensive and quantitative profile of HRMs in
vascular endothelial cells (ECs). Bioinformatics in conjunction
with experimental validation revealed that Let-7 and miR-103/107
are robustly induced in ECs and consequently target AGO1. Subse-
quently, a translational desuppression pathway upregulates VEGF
and enhances angiogenesis in vitro and in vivo. In addition, results
from clinical specimens provided evidence that this pathway may
be implicated in tumor angiogenesis and progression. These find-
ings suggest a novel role of HRMs in hypoxia-induced gene expres-
sion and the consequent angiogenesis via targeting AGO1.
Results
Hypoxia induces Let-7 and miR-103/107. We performed SBS deep
sequencing of small RNA libraries derived from HUVECs subjected
to normoxia or hypoxia. Among all the miRNAs profiled, Let-7 and
miR-103/107 were highly induced miRNA families (Table 1 and
Supplemental Table 1; supplemental material available online with
this article; doi:10.1172/JCI65344DS1). Their induction by hypoxia
was confirmed by Northern blotting (Figure 1A). The marked induc-
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. doi:10.1172/JCI65344.
2. research article
2 The Journal of Clinical Investigation http://www.jci.org
tion of specific miRNA paralogs, namely, Let-7a, Let-7e, miR-103,
and miR-107, was validated by TaqMan quantitative PCR (qPCR)
(Figure 1B). Because HIF1α is the key TF that mediates the induc-
tion of many hypoxia-inducible genes and was previously reported
to induce miR-103 in cancer cells (18), we examined whether HIF1α
can upregulate these HRMs in ECs. As depicted in Table 2 and Sup-
plemental Table 2, Let-7 and miR-103/107 genes were all predicted
to contain 1 or more conserved HREs in their promoter regions.
Indeed, overexpression of HIF1α in HUVECs confirmed the induc-
tion of Let-7a, Let-7e, miR-103, and miR-107 (Figure 1C).
Let-7andmiR-103/107targetAGO1.GiventhatLet-7andmiR-103/107
are greatly induced by hypoxia, we predicted the functional targets
of these miRNAs by using miRanda (20), RNAhybrid (21), and Tar-
getScan (22). Let-7 and miR-103/107 were predicted to target 169
and 148 mRNAs, respectively; these genes included AGO1, homeo-
box protein A1 (HOXA1), and apoptosis-antagonizing TF (AATF)
(Supplemental Table 3, A and C). To verify whether the predicted
genes are bona fide targets of HRMs, we first tested whether their
expression was downregulated by hypoxia. The expression of AGO1
and HOXA1 but not AATF was decreased and that of HIF1α was
increased in ECs under hypoxic stress (Figure 2A). Because AGO1
is a key protein anchoring miRISC and its mRNA 3′ UTR contains
conserved seed sequences for all HRMs (Supplemental Figure 1), we
further explored the HRM targeting of AGO1. Overexpression of
the pre-miRs of Let-7 or miR-103/107 decreased the level of AGO1
in ECs under normoxia. In contrast, the inhibition of Let-7 or
miR-103/107 with locked nucleic acid (LNA) increased the expres-
sion of AGO1 in hypoxic HUVECs (Figure 2, D and E, and Supple-
mentalFigure2B).Furthermore,exogenouslyexpressedHIF1α,sim-
ilartohypoxiaorpre-miRs,suppressedthelevelofAGO1(Figure2F).
These data suggest that HIF1α-induced Let-7 and miR-103/107
suppressed AGO1 expression under hypoxia.
HRMs targeting AGO1 mRNA in miRISC. To demonstrate a direct
targeting of AGO1 3′ UTR by Let-7 and miR-103/107, we created
reporter constructs containing luciferase fused to the WT AGO1
3′ UTR (AGO1–3′ UTR). Human embryonic kidney 293 (HEK293)
cells cotransfected with the AGO1–3′ UTR reporter together with
pre–Let-7e, pre–miR-103, or a combination of these 2 pre-miRs
conferred lower luciferase activity as compared with cells cotrans-
fected with control RNA (Figure 3A). However, pre–Let-7e or pre–
miR-103 induction was unable to decrease the luciferase activity
in cells cotransfected with AGO1–3′ UTR mutant constructs with
mutated Let-7 or miR-103/107 binding sites. We also constructed
an AGO2–3′ UTR reporter. The luciferase reporter activity of the
constructed AGO2–3′ UTR was not affected by the cotransfected
pre–Let-7 or miR-103, which is consistent with the lack of Let-7 and
miR-103/107 target sequences at the AGO2–3′ UTR. Furthermore,
hypoxia, like pre–Let-7 or miR-103, decreased the luciferase activity
of AGO1–3′ UTR, but not that of AGO2–3′ UTR in ECs (Figure 3B).
miRNA/mRNA complexes associate with AGO proteins to
form miRISC. For AGO1–4 found in human cells (13), hypoxia
decreased only AGO1 levels in HUVECs (Figure 3C; AGO4 pro-
tein level was under the detection limit), but the mRNA level of
AGO1–3 remained unchanged (Figure 3D). These results suggest
that HRMs targeting AGO1 was due mainly to translational sup-
pression but not AGO1 mRNA degradation, possibly because of
imperfect complementarities between the Let-7 and miR-103/107
and AGO1 mRNA (refs. 23, 24 and Supplemental Figure 1).
Let-7 and miR-103/107 should target AGO1 mRNA within the
AGO1-associated miRISC. We therefore compared the amount of
AGO1-associated Let-7, miR-103/107, and AGO1 mRNA in nor-
Table 1
HRM profiles from SBS deep sequencing
miRNA Reads Reads Fold
(normoxia) (hypoxia) change
Let-7a 277 2614 6.96
Let-7b 146 900 4.54
Let-7c 205 1089 3.92
Let-7d 252 1032 3.02
Let-7e 52 655 9.29
Let-7f 984 5820 4.36
Let-7g 171 1476 6.36
Let-7i 142 488 2.53
miR-103 334 3743 8.26
miR-107 336 3789 8.31
Figure 1
Hypoxia induces Let-7 and miR-103/107 in ECs. (A and B) HUVECs were kept under normoxia (21% O2) or were subjected to hypoxia (2% O2)
for 24 hours. (A) Northern blotting analysis of Let-7 and miR-103/107 expression. 18S rRNA was detected as loading control. (B) The indicated
miRNAs were detected by TaqMan qPCR.The relative expression was normalized to that of U6 RNA, with that of normoxia set as 1. (C) HUVECs
were infected with Ad-HIF1α for 72 hours, and miRNAs were quantified by TaqMan qPCR.The relative level of miRNA expression was compared
with that of the mock-infected group (0 MOI), set as 1. Bar graphs represent mean ± SD from 3 independent experiments. *P < 0.05 compared
with normoxia in B or mock-infected controls in C.
3. research article
The Journal of Clinical Investigation http://www.jci.org 3
moxic HUVECs and that in hypoxic cells. IP of AGO1 and sub-
sequent qPCR analysis revealed that Let-7a/e and miR-103/107
were significantly enriched in AGO1-miRISC in hypoxic ECs, even
though the level of AGO1 was lower (Figure 3, E and F, and Sup-
plemental Figure 3). Consistently, the level of AGO1 mRNA, as an
HRM target, was also increased in the AGO1-miRISC in hypoxic
cells (Figure 3G). However, we detected neither miRNA nor AGO1
mRNA in IgG isotype controls (Supplemental Figure 3).
Translational desuppression of VEGF mediated by HRMs targeting AGO1.
ThesequentialeventsofHIF1αupregulation,Let-7andmiR-103/107
induction, and AGO1 suppression (Figure 4, A–C) led us to propose
an HRM-induced translational desuppression pathway: during
hypoxia, the HIF1α-induced HRMs, through repressing AGO1-
miRISC, release mRNAs that are translationally suppressed under
normoxia, which results in the upregulation of the encoded proteins
(Figure 4D). To validate this pathway, we searched for mRNA that
is decreased in AGO1-miRISC under hypoxia. We tested VEGF as a
prototypicgenebecauseitcanbeinducedbyhypoxiaduetoincreased
mRNA stability or translational efficiency, possibly mediated by
AGO1-miRISC.Indeed,wedetectedamarkeddecreaseinmRNAlevel
of AGO1-associated VEGF in ECs under hypoxia as compared with
the normoxic control (Figure 4E). We then tested whether HRM tar-
geting AGO1 mediates hypoxia-induced VEGF. Inhibition of HRMs
or overexpression of AGO1 by using a construct encoding the AGO1
ORF without its 3′ UTR attenuated the hypoxia-induced VEGF in
ECs (Figure 4, F and G). However, when AGO1 was knocked down
to mimic hypoxia suppression, VEGF expression was increased in
normoxic ECs. Furthermore, when HUVECs were treated with cyclo-
heximide, a translation inhibitor, the effect of AGO1 knockdown on
VEGF induction was abolished (Figure 4H). In a control experiment,
VEGF levels were not altered by siRNA against AGO2, whose expres-
sion was not repressed under hypoxia (Supplemental Figure 4A).
These results suggest that hypoxia-induced VEGF requires transla-
tional desuppression through HRM-specific targeting of AGO1.
HRMs targeting AGO1 enhances angiogenesis. To investigate the
functional consequence of translational desuppressed VEGF, we
assessed the role of HRMs targeting of AGO1 in in vitro angio-
genesis. Consistent with results presented in Figure 4, F–H, block-
age of HRMs abolished the hypoxia-induced endothelial tube
formation in Matrigel (Figure 5A), which was simulated by AGO1
overexpression (Figure 5B). In contrast, knockdown of AGO1 in
normoxic ECs resulted in enhanced tube formation (Figure 5C).
These findings indicate that HRMs targeting AGO1 is functionally
important for angiogenesis in vitro.
To validate these findings in vivo, we first examined the expression
of HRMs and AGO1 in C57BL/6 mice subjected to 5-day 10% O2
treatment, which mimics the partial pressure of oxygen at approxi-
mately 5000 m altitude (25). Let-7a, Let-7e, and miR-103 expression
were abundant and robustly induced in multiple tissues/organs
in mice exposed to such hypoxia, whereas miR-107 expression was
less abundant and not significantly changed (Figure 5D). In accor-
dance with the miRNA induction, AGO1 expression was suppressed
(Figure5E).Similarresultswereobtainedinmicesubjectedtohypox-
ia with lower oxygen content or longer duration (8% O2 for 3 days or
10% O2 for 7 days; Supplemental Figure 5, A–C). We next delivered
antagomirs against Let-7a, Let-7e, and miR-103 to the hind limbs of
C57BL/6 mice (Figure 5F): Hypoxia-induced VEGF expression was
attenuated in antagomir-administered muscles as compared with
muscles of mice receiving control RNA (Figure 5G).
ToevaluatetheeffectofmiRNAtargetingofAGO1onangiogenesis
invivo,weimplantedMatrigelmixedwithcontrolRNAorantagomirs
against Let-7a, -7e, and miR-103 in C57BL/6 mice. Hypoxia-induced
angiogenesiswasapparentinMatrigelplugsmixedwithcontrolRNA,
assignifiedbytheincreasedhemoglobin(Hb)content,infiltratedred
blood cells, and staining for vWF and CD31 (Figure 6, A–C). Notably,
these angiogenic responses were greatly decreased in Matrigel plugs
mixedwithantagomirs,whichabolishedthehypoxia-inducedLet-7a,
-7e, and miR-103 (Figure 6, A–C, and Supplemental Figure 6). In nor-
moxic mice, little angiogenesis was seen in Matrigel plugs mixed with
controlRNAorantagomirs.WealsotestedtheroleofAGO1byusing
SCID mice. Matrigel mixed with HUVECs was s.c. injected into the
dorsal sides of SCID mice. As compared with HUVECs transfected
with a control vector, cells overexpressing AGO1 resulted in a lower
angiogenic response in hypoxic mice (Figure 6, D and E). In the com-
plementary experiment, we administered Matrigel mixed with ECs
transfected with control RNA or AGO1 siRNA to SCID mice. Under
normoxia, angiogenesis was increased in the implanted Matrigel
plugs, which depended on the dose of AGO1 siRNA (Figure 6, F–H).
Thus,angiogenesiscouldbeinducedbyHRMstargetingAGO1,lead-
ing to translationally desuppressed VEGF.
The HRM/AGO1/VEGF pathway is correlated with tumorigenesis.
Because hypoxia-induced angiogenesis is critical in tumorigenesis,
we postulated that HRM-mediated desuppression is also present
in solid tumors. We therefore examined the levels of miRNAs and
AGO1 in breast tumor xenografts in SCID mice. In these expanded
xenografts, hypoxic cores were formed (Figure 7A). As compared
with oxygenated distal regions, the hypoxic core showed increased
HRM levels but decreased AGO1 expression (Figure 7, B and C).
Given that hypoxic stress is associated with the majority of human
tumors (26), we compared HRM levels in paired tumor and normal
tissuesfrompatientswithbreast,colon,orlivercancers.Let-7eand/or
miR-103levelswereelevatedintumortissuesin11of16pairs(Figure
7D). To evaluate the clinical relevance of the AGO1-mediated trans-
lational desuppression of VEGF, we performed in situ hybridization
(ISH) and immunohistochemistry (IHC), and correlated the VEGF
andAGO1expressionin173humanhepatocellularcarcinoma(HCC)
specimens. We found inverse correlations between HRM and AGO1
expression, AGO1 and VEGF expression, and AGO1 and microvessel
density (revealed by CD31 staining) (Figure 7, E and F, and Supple-
mental Table 4). Furthermore, a high level of AGO1 but low level of
VEGF was associated with both increased disease-free (P= 0.009) and
Table 2
Putative HREs and HIF1α-binding sites in the HRM promoter
regions
miRNA HRE Strand Binding sequence
Let-7a-1 346 + ACAGACGTGTTGAT
Let-7a-3 –2130 + CCTGACGTGAGTTC
Let-7b –2130 + CCTGACGTGAGTTC
Let-7d –1086 + GAATACGTGCCGCC
Let-7e –2363 + GCAAACGTGGCAAA
Let-7f-1 356 + ACAGACGTGTTGAT
Let-7f-2 –345 – GACGACGTGGGAAG
Let-7g –159 – CGAGACGTGCAGGG
Let-7i 290 + GGTCACGTGGTGAG
miR-103-1 –669 – GACCACGTGCAATT
miR-103-2 –2192 + CCCAACGTGCTGGG
miR-107 –475 + CACCACGTGATGCC
4. research article
4 The Journal of Clinical Investigation http://www.jci.org
overall survival (P = 0.055). In contrast, poor cancer survival was cor-
related with low levels of AGO1 but high levels of VEGF (Figure 7,
G and H). These findings suggest that the HRM-targeted AGO1 and
the subsequent desuppressed VEGF expression may be involved in
tumor progression and is indicative of cancer prognosis.
Discussion
Under hypoxia, cells adjust their transcription and translation to
adapt to the deficit in O2 and energy. Hypoxia decreases global
protein synthesis, mainly by phosphorylation of eukaryotic initia-
tion factor 2 α and inhibition of mammalian target of rapamycin
activity, thus causing suppressed 5′-cap-dependent mRNA trans-
lation (27–29). However, many genes involved in glycolysis, cell
cycle arrest, and angiogenesis are highly expressed. The transcrip-
tional induction by TFs such as HIF ensures an abundant level of
mRNAs that enables the translation of some of these genes under
the suppressed translational machinery. In addition to direct
transcriptional induction, noncanonical IRES-initiated transla-
tion and decreased proteosome-mediated protein degradation
are involved in the induction of some of these hypoxia-inducible
genes (7, 30, 31). Our current study suggests that HRMs such as
Let-7 and miR-103 targeting AGO1 results in translational desup-
pression under hypoxia. This newly defined mechanism may
allow for a timely release of sequestered mRNAs for translation,
which synergizes with those previously defined, thus leading to
an optimal concentration of mRNAs to overcome the suppressed
translational machinery.
In the current study, we identified VEGF, a key mediator of
hypoxic response, as a target gene regulated by such machinery.
We provided evidence that HRMs, by targeting AGO1, release
VEGF mRNA sequestered in miRISC, thus contributing to VEGF
induction (Figure 4, E–H). Because the silencing effect of miRISC
requires AGO1 protein as well as the miRNA-mRNA duplex,
hypoxia-suppressed AGO1 expression should also release miRNAs
that would otherwise target VEGF mRNAs under normoxia.
Indeed, hypoxia greatly decreased AGO1-associated miR-15 and
miR-29 levels, previously reported to target VEGF (refs. 32, 33,
and Supplemental Figure 7A). In contrast, the mRNAs encoding
hypoxia-suppressed genes and their cognate miRNAs would still
be enriched in hypoxic AGO1-miRISC. For example, the AGO1
protein-associated AGO1 mRNA and HRMs were concomitantly
higher under hypoxia, despite the reduced expression of AGO1
(Figure 3, E–G). Of note, HIF2α overexpression can also induce
HRMs, probably through the putative HREs in the promoter
regions of these HRMs (Supplemental Figure 8). Therefore, both
HIF1α and HIF2α may upregulate HRMs under hypoxia. Given
the induction of HRMs by HIF (Figure 1C and Supplemental Fig-
ure 8) and the time course of HIF, HRM, and AGO1 expression
Figure 2
Let-7 and miR-103/107 target AGO1. (A) HUVECs were subjected to normoxia (time 0) or hypoxia for the indicated times. Cell lysates underwent
SDS-PAGE, then Western blot analysis with various antibodies. (B and C) HUVECs were transfected under normoxia for 72 hours with control
RNA, pre–Let-7a and pre–Let-7e, or pre-103 and pre-107, at 20 or 40 nM each.Western blot analysis was performed to assess the AGO1 level. (D
and E) HUVECs were transfected with LNAs inhibiting Let-7a and Let-7e or miR-103 and miR-107 at indicated concentrations for 48 hours before
24-hour hypoxic stress. Western blot analysis was performed to assess the level of AGO1. (F) Western blot analysis of AGO1 level in HUVECs
infected with Ad-HIF1α. Relative protein expression was obtained by normalizing to that of β-actin or α-tubulin. Bar graphs represent mean ± SD
from 3 independent experiments. *P < 0.05 versus respective controls set to 1.
5. research article
The Journal of Clinical Investigation http://www.jci.org 5
(Figure 2A and Figure 4, A–C), the translational desuppression
may also be considered an HIF-integrated network. Such a mode
of action, combined with enhanced transcription and translation,
may confer maximal upregulation of VEGF and serve as an essen-
tial mechanism, especially in chronic hypoxia. In line with this, the
hypoxia-activated HRM/AGO1 pathway was detected under both
physiological and pathophysiological conditions in vivo (Figure 5,
D and E, and Figure 7, A–C).
Because of the multitude of genes regulated by miRNAs and
miRISC, other hypoxia-inducible genes may also be translation-
ally desuppressed in this manner. To test this likelihood, we per-
formed microarray experiments to compare mRNAs associated
with AGO1-miRISC under normoxia and hypoxia. In addition
to VEGF, multiple genes involved in glycolysis and angiogen-
esis may be desuppressed under hypoxia (Supplemental Tables
5 and 6). Among these, mRNAs encoding PDGF and placen-
tal growth factor (PlGF) were verified by qPCR (Supplemental
Figure 7B). Belonging to the same family as VEGF, PDGF and
PlGF are highly induced by hypoxia and are potent angiogenic
factors under various physiological and pathological conditions
(34–36). Interestingly, consensus HREs were not found in the
promoter regions of PDGF and PlGF. Hence, their induction by
hypoxia seems independent of HIF direct transactivation (37).
An intriguing issue is the specificity of AGO1-mediated desup-
pression because not all mRNA transcripts were released from
the AGO1-miRISC under hypoxia (Figure 3G). This finding sug-
gests that the desuppression mechanism is not a generic effect
resulting from global AGO1 decrease.
Among all the AGO family members, only AGO1 is suppressed
by hypoxia in ECs. A recent study reported that hypoxia increases
AGO2 expression through posttranslational hydroxylation in cul-
tured SMCs, which enhances its stability (38). In contrast, Ho et al.
reported that hypoxia slightly decreases AGO2 in ECs (39). How-
ever, none of our newly identified HRMs were predicted to target
AGO2, nor did hypoxia significantly alter AGO2 expression in
cultured ECs or various tissues in vivo (Figure 3, A–D, and Supple-
mental Figure 5D). Because AGO1 expression is decreased under
hypoxia, AGO2 is likely involved in mediating miRNA targeting.
Indeed, AGO2-associated HRMs were also increased under hypox-
ia (Supplemental Figure 4B). In addition, AGO2 would be required
for stabilizing mature miRNAs under hypoxia (40). Because levels
of AGO2-associated VEGF, PlGF, and PDGF mRNAs in normoxic
cells were similar to those in hypoxic cells (Supplemental Figure
4C) and AGO2 silencing did not enhance VEGF expression in ECs
(Supplemental Figure 4A), AGO2 may not play a major role in the
hypoxia-induced translational desuppression.
Results presented in Figure 7 suggest that HRM/AGO1-mediated
translational desuppression may contribute to hypoxia-induced
Figure 3
Posttranscriptional targeting of AGO1 mRNA in AGO1-mediated miRISC. (A) HEK293 cells were transfected with the WT Luc-AGO1–3′ UTR
(WT), Luc-AGO1–3′ UTR with miR-103/107 or Let-7 target sites mutated (mut), or Luc-AGO2–3′ UTR, together with pre–Let-7e (40 nM), pre-103
(40 nM), pre–Let-7e and pre-103 (20 nM each), or control RNA (40 nM). (B) Bovine aortic ECs (BAECs) transfected with Luc-AGO1–3′ UTR or
-AGO2–3′ UTR were subjected to normoxia (Nx) or hypoxia (Hx). CMV–β-gal was cotransfected in all groups as a transfection control. Luciferase
activity was normalized to that of β-gal. (C–G) HUVECs were subjected to normoxia or hypoxia. (C and D) Western blot and qPCR analyses of
protein and mRNA levels of AGO1–3. (E–G) AGO1 was immunoprecipitated from cell lysates. The immunoprecipitates were subjected to AGO1
immunoblotting (F) and the AGO1-associated miRNAs and AGO1 mRNA were quantified by qPCR (E and G). Data represent mean ± SD from 3
independent experiments. *P < 0.05 compared with control RNA group in A or normoxia group in B–G.
6. research article
6 The Journal of Clinical Investigation http://www.jci.org
angiogenesis during tumorigenesis. Antagonists against this angio-
genic pathway may disrupt the tumorigenesis by targeting VEGF
and other proangiogenic factors. Because of the heterogeneity of
solid tumors and the distinct tumor origin, the induced HRMs
and the involved mechanisms may differ. For example, although
hypoxia can decrease AGO1 expression via Let-7 targeting, Let-7
may also target oncogenes (41, 42). The overall functional outcome
of Let-7 would depend on integrated targeting of genes. In contrast
to Let-7’s role as a tumor suppressor, miR-103/107 expression was
found inversely correlated with breast cancer survival (43). We and
our collaborators also demonstrated that miR-103/107 levels are
negatively correlated with survival rates for colon cancer patients
(44). Thus, during the development of certain types of cancer,
miR-103/107 but not Let-7 may be protumorigenic.
Interestingly, Let-7 and miR-103/107 also target human Dicer
(43, 45). As well, recent work by Ho et al. demonstrated that full
expression of hypoxia-responsive genes such as VEGF in chronic
hypoxia depends on hypoxia-repressed Dicer and changes in post-
transcriptional gene regulation (39). Together with Dicer-mediated
suppression of miRNA targeting, HRM/AGO1-mediated transla-
tional desuppression adds a new mode of regulation of hypoxia-
induced gene expression in concert with the HIF-orchestrated net-
work. These pathways highlight the importance of miRNAs and
their machinery in modulating the cellular response to hypoxic
stresses. Collectively, our findings demonstrate the crucial role of
HRM/AGO1-mediated translational desuppression in the hypoxic
response (Figure 4D and Figures 5 and 6) and the therapeutic
potential of targeting this pathway in pathological angiogenesis.
Methods
Small RNA library and SBS deep sequencing. Total RNA was extracted by a stan-
dard Trizol protocol (Invitrogen). The small RNA library was constructed
as described (46). Briefly, 20- to 30-nt small RNAs were gel isolated; then the
5′ and 3′ ends were ligated to single-stranded oligonucleotides (5′ tags and
3′ adaptor) (for oligo sequences, see Supplemental Methods). The ligates
then underwent RT-PCR, which resulted in a library of small RNA cDNA
molecules. After performing quality control, we used the cDNA library for
deep sequencing by Illunima analyzer. miRNA profiles are demonstrated by
their reads in the libraries derived from normoxic or hypoxic cells, and fold
change under hypoxia relative to normoxic controls was thus determined.
In silico bioinformatics analysis. Reads from SBS deep sequencing were ana-
lyzed as follows: (a) sorting of normoxia and hypoxia libraries based on 5′
Figure 4
Translational desuppression of VEGF caused by AGO1 targeting. (A–C) HUVECs were subjected to normoxia or hypoxia for the indicated times.
Western blot analysis of protein levels of HIF1α (A) and AGO1 (C) and TaqMan qPCR analysis of miRNAs (B). (D) Model of translational suppres-
sion under normoxia and desuppression under hypoxia. Red arrows indicate up- or downregulation of various molecules. (E) AGO1-associated
mRNAs were enriched by IP. Level of VEGF was detected by qPCR. (F) HUVECs were transfected with a mixture of anti–Let-7a/e/miR-103
(10 nM each) or control RNA (30 nM). (G) HUVECs were transfected with a control vector expressing HA or HA-AGO1-ORF plasmid containing
AGO1 ORF without 3′ UTR. (H) HUVECs were transfected with AGO1 siRNA or control RNA for 48 hours, then treated with vehicle control or
cycloheximide (CHX) (5 μg/ml) for 4 hours. Cells in F–H were then subjected to hypoxia or kept as normoxia controls, and various proteins were
detected by Western blot analysis. *P < 0.05 compared with normoxia group.
7. research article
The Journal of Clinical Investigation http://www.jci.org 7
Figure 5
HRM/AGO1 pathway mediates hypoxia-induced angiogenesis in vitro and responds to hypoxia in vivo. (A–C) HUVECs were prepared as
described in Figure 4, F–H, and underwent in vitro angiogenesis assays.The tube/joint numbers were counted, with normoxia/control RNA group
(A), normoxia/control plasmid group (B), or control RNA group (C) set to 1. Scale bar: 100 μm. (D and E) C57BL/6 mice were subjected to hypoxia
(10% O2) or normoxia (21% O2) for 5 days. Let-7 and miR-103/107 expression in multi-organs/tissues was detected by TaqMan miRNA qPCR
(D) and AGO1 protein level by Western blot analysis (E). The levels of miRNAs in normoxia were set to 1. (F and G) Control RNA or antagomirs
(antmiR) were delivered with pluronic gel to hind-limb muscles of C57BL/6 mice and then kept under normoxia or hypoxia for 5 days.The miRNAs
in the hind limbs were quantified by qPCR (F) and VEGF by Western blot analysis (G). (D–G) Data represent results from at least 6 animals per
group. *P < 0.05 compared with controls or between indicated groups.
9. research article
The Journal of Clinical Investigation http://www.jci.org 9
administered into each designated hind limb. After immediate gelification,
the skin was reapproximated. The animals were kept for 2 days for recovery
before subsequent procedures.
In vivo Matrigel plug assay followed a published protocol (52) with
modification. First, 2 Matrigel plugs premixed with 60 U/ml heparin and
40 ng/ml basic fibroblast growth factor, together with 15 μg control RNA
or antagomirs (5 μg each of Let-7a, Let-7e, and miR-103) were s.c. injected
(500 μl) into the dorsal surface of C57BL/6 mice. After 24-hour recovery,
animals were subjected to normoxia or hypoxia for 5 days. The mice were
sacrificed, and Matrigel plugs were photographed and harvested for Hb
quantification and histology. For Hb quantification, Matrigel plugs were
homogenized in 200 μl distilled deionized water. The supernatant after
centrifugation was used for Drabkin’s assay (Sigma-Aldrich). For IHC,
Matrigel plugs were fixed with formaldehyde and processed as described
in Supplemental Methods.
The experiments involving SCID mice were conducted as described (53),
with modification. Briefly, HUVECs were transfected with siRNA or DNA
plasmids. At 18 hours after transfection, 1 × 106 cells were mixed with 400 μl
Matrigel containing 30 units/ml heparin and s.c. injected into the dor-
sal surface of 8-week-old female SCID mice. On day 3 (for AGO1 rescue
experiments) or 5 (for AGO1 siRNA experiments), mice were sacrificed and
Matrigel plugs were harvested and photographed.
Female SCID mice (6 to 8 weeks old) were used as recipients in the tumor
xenograft model. Specifically, MDA-MB-231 human breast adenocarcino-
ma cells (5 × 105) were injected into the mammary fat pad. Body weight and
tumor volume were monitored weekly for 4 weeks. Tumors of 10–15 mm
in diameter were collected at autopsy. Core regions of necrotic tumors and
distal areas were separated for detection of miRNA and AGO1 protein.
Clinical samples, ISH, and IHC. The surgical specimens used for ISH and
IHC were formalin-fixed and paraffin-embedded (FFPE) before being
archived. ISH and IHC were performed following protocols previously
described (44) with minor modification. For ISH, 50 nM 3′-DIG–labeled
Let-7e or miR-103 LNA were used as probes. Upon reaction with alka-
line phosphatase-linked antibody against DIG (Roche), ISH signals were
visualized by use of bromochloroindolyl phosphate-nitro blue tetrazo-
lium substrates. For IHC, rabbit antibodies against human AGO1 (Cell
Signaling), VEGF (Santa Cruz Biotechnology Inc.), or CD31 (Santa Cruz
Biotechnology Inc.) were used as primary antibodies, followed by sequen-
tial incubation with biotinylated secondary antibody, and horseradish
peroxidase-conjugated streptavidin. The staining was visualized by use of
3-3′ diaminobenzidine tetrahydrochloride (DAB), and counterstaining was
with light hematoxylin. All samples were viewed and photographed under
an Olympus microscope. A 4-point scoring system based on staining inten-
sity (range from 0 to 3, with 0 being no expression and 3 maximal expres-
sion) was used to evaluate the relative expression of various molecules. All
IHC images were reviewed and scored independently by 2 pathologists.
Statistics. The significance of variability was determined by 2-tailed Stu-
dent’s t test for 2-group comparison. For multi-group comparison, ANOVA
was performed, followed by post-hoc testing to compare differences among
groups. Unless otherwise indicated, all results are presented as mean ± SD
or mean ± SEM from at least 3 independent experiments. P < 0.05 was
considered statistically significant. Correlation between prognostic mol-
ecules was determined by Spearman’s nonparametric correlation test. The
Kaplan-Meier estimate was used for analysis of disease-free and overall sur-
vival, with the log-rank test used to compare the difference.
Study approval. The animal experimental protocols were approved by the
Institutional Animal Care and Use Committee (IACUC) of the Univer-
sity of California, Riverside, and the University of California, San Diego.
The normal and tumor (N/T) paired samples and human tissues used for
miRNA ISH and IHC were from patients undergoing tumor resection or
tag sequences, (b) trimming the 3′ adaptor sequences, and (c) eliminat-
ing sequences of less than 18 or more than 28 nt. All obtained sequences
were searched against the human genome by use of a short oligonucleotide
alignment program for completely matched sequences. After alignment to
the miRNA registry (miRBase 13.0), the miRNA expression profiles were
generated and presented as specific miRNA with copy numbers normalized
by total small RNA reads. HREs in miRNA promoter regions were searched
as described (47). Briefly, 3 binding consensus sequences from proTrans-
Fac 7.0 (48) were used to scan the putative promoter regions, and potential
HREs were calculated by the position-weight matrix approach. To predict
the miRNA targets, we used miRanda, RNAhybrid, and TargetScan to scan
cross-species–conserved 3′ UTRs of mRNAs.
Plasmids and luciferase assay. Human AGO1 or AGO2 3′ UTR contain-
ing miR-103/107 and Let-7 target sequences were subcloned into pMIR-
REPORT vector (Ambion) to generate pMIR-Luc-AGO1/2–3′ UTR. To cre-
ate the pMIR-Luc-AGO1–3′ UTR-103/7-mut, a GC to CG mutation was
introduced into the miR-103/107 target sequence by using PfuTurbo (Strat-
agene). To create the pMIR-Luc-AGO1–3′ UTR–Let-7-mut, approximately
50-bp flanking Let-7 target sites were deleted using a 2-step PCR deletion
strategy (49). N-terminal HA-AGO1-ORF (plasmid 21535; Addgene) was
originally generated by Lian et al. (50). Luciferase reporter constructs were
cotransfected with β-gal with or without pre-miR or control RNA. Lucifer-
ase activity was measured by use of the Dual-Glo Luciferase Reporter Assay
Kit (Promega). The luciferase activity was normalized to that of β-gal.
Immunoprecipitation of AGO-miRISC. HUVECs were lysed with a buffer con-
taining 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA,
and100units/μlRNaseinhibitor.Thelysateswereincubatedwithanti-AGO1
(clone 2A7; Wako Chemicals) or anti-AGO2 (clone 4G8; Wako Chemicals) at
4°CovernightandthenproteinGDynabeadsfor4hours.Thebeadswerethen
spun down, and the immunoprecipitated proteins and RNAs were extracted
with lysis buffer and Trizol, respectively. The microarray data of AGO1-
associated mRNAs have been deposited in Gene Expression Omnibus (GEO
GSE43165), with an assigned accession number upon approval of GEO.
Animal experiments. We used 8- to 12-week-old male C57BL/6 or female
SCID mice. The mice subjected to hypoxia were placed in a sealed chamber
infused with 10% O2 and 90% N2 that was kept at room temperature for
5 days. Age- and sex-matched mice were maintained at room temperature,
which represented the normoxic control.
AntagomiRs against Let-7a, -7e, and miR-103 were designed as described
(51) (see Supplemental Methods for details). On day 1, mice were anesthe-
tized. Then 30 μg control RNA or antagomirs (10 μg of Let-7a, Let-7e, and
miR-103 each) mixed with 25% pluronic gel in a total volume of 80 μl was
Figure 6
HRM/AGO1 pathway regulates hypoxia-induced angiogenesis in vivo.
(A–C) Matrigel mixed with control RNA (15 μg/plug) or antagomirs
against Let-7a, Let-7e, and miR-103 (5 μg/plug each) was injected
s.c. into C57BL/6 mice before 5-day normoxia or hypoxia. Gross
morphology (A) and Hb quantification (B) as well as H&E, vWF, and
CD31 staining of Matrigel plugs (C). (D and E) HUVECs transfected
with control or HA-AGO1-ORF plasmid were mixed with Matrigel and
s.c. injected into SCID mice, which were then kept under normoxia or
hypoxia for 3 days. Gross morphology (D) was photographed and Hb
quantified (E). (F–H) HUVECs transfected with control RNA (40 nM)
or AGO1 siRNA (20 or 40 nM) were mixed with Matrigel and s.c. inject-
ed into SCID mice. Five days after injection, the plugs were harvested
for gross morphology (F), Hb quantification (G), histology, and IHC
(H). In C, E, and G, the Hb content in normoxia/control RNA or con-
trol plasmid group was set to 1. Bar graphs represent mean ± SEM.
Results are from 5–10 animals per group. Scale bar: 100 μm. *P < 0.05
compared with controls or between indicated groups.
10. research article
10 The Journal of Clinical Investigation http://www.jci.org
Figure 7
Implication of HRM-mediated VEGF desuppression in tumorigenesis. (A–C) Mammary fat pads of SCID mice were implanted with 5 × 105
MDA-MB-231 human breast cancer cells.After 4 weeks, mice were killed and tumors were harvested. Core regions of necrotic tumors and distal
areas shown in A were collected for detection of miRNAs (B) and AGO1 protein (C). Scale bar: 2 mm. (B) Specimens collected from 10 animals
were pooled into 4 samples (each containing 2 or 3 specimens as indicated) for miRNA qPCR. Bars represent the ratio of miRNA level in the core
relative to that in the distal region. (C) Representative images of Western blot analysis with samples from individual mice. (D) Levels of Let-7e
and miR-103 was detected in paired normal (N) and tumor (T) tissues, and the T/N expression ratio was calculated. (E and F) Representative IHC
staining of AGO1, VEGF, CD31, and ISH of Let-7e and miR-103 in serial sections from patients with AGO1 high/VEGF low (E) and AGO1 low/
VEGF high expression patterns (F). Scale bar: 200 μm (×100); 100 μm (×400).Yellow arrowheads indicate the location of microvessels. (G and H)
Kaplan-Meier analysis of disease-free (G) and overall survival (H) for 173 HCC patients, stratified by AGO1 and VEGF expression.
11. research article
The Journal of Clinical Investigation http://www.jci.org 11
(Department of Oral Biology, University of Florida College of Den-
tistry Health Science Center) for sharing the HA-AGO1 plasmid in
theAddgenerepository.Theauthorsaregratefulforusefulcomments
from David Johnson, Craig Byus, Amy Pasquinelli, and Traci Marin.
Received for publication June 15, 2012, and accepted in revised
form January 3, 2013.
Address correspondence to: John Y-J. Shyy, 1126 Webber Hall,
900 University Ave., Riverside, California 92521, USA, or 9500
Gilman Dr., La Jolla, California 92093-0726, USA. Phone:
951.827.3863 or 858.534.2878; Fax: 951.827.5504; E-mail: john.
shyy@ucr.edu and jshyy@ucsd.edu.
surgical procedures at Taipei Veterans General Hospital. All clinical sam-
ples were obtained with informed consent and institutional review board
approval (protocol number: 201010021IC) and paired based on patient
sex, age, and histopathological diagnoses.
Supplemental methods. Additional experimental procedures are described
in Supplemental Methods.
Acknowledgments
This work is supported in part by the NIH Heart, Lung, and Blood
Institute (grants HL89940, HL105318, and HL106567 to J.Y-J. Shyy),
the American Heart Association (grant 11POST7590128 to Z. Chen),
and the National Science Council of the Republic of China (grant
NSC 101-2911-I-009-101 to H. Huang). We thank Edward Chan
1. Manalo DJ, et al. Transcriptional regulation of vas-
cular endothelial cell responses to hypoxia by HIF-1.
Blood. 2005;105(2):659–669.
2. Semenza GL. Hypoxia-inducible factor 1 (HIF-1)
pathway. Sci STKE. 2007;2007(407):cm8.
3. Wenger RH, Stiehl DP, Camenisch G. Integration
of oxygen signaling at the consensus HRE. Sci
STKE. 2005;2005(306):re12.
4. Kenneth NS, Rocha S. Regulation of gene expres-
sion by hypoxia. Biochem J. 2008;414(1):19–29.
5. Arany Z, et al. HIF-independent regulation of VEGF
and angiogenesis by the transcriptional coactivator
PGC-1alpha. Nature. 2008;451(7181):1008–1012.
6. Forsythe JA, et al. Activation of vascular endothelial
growth factor gene transcription by hypoxia-induc-
ible factor 1. Mol Cell Biol. 1996;16(9):4604–4613.
7. Stein I, Itin A, Einat P, Skaliter R, Grossman Z, Kesh-
et E. Translation of vascular endothelial growth fac-
tor mRNA by internal ribosome entry: implications
for translation under hypoxia. Mol Cell Biol. 1998;
18(6):3112–3119.
8. Ikeda E, Achen MG, Breier G, Risau W. Hypoxia-
induced transcriptional activation and increased
mRNA stability of vascular endothelial growth
factor in C6 glioma cells. J Biol Chem. 1995;
270(34):19761–19766.
9. Ray PS, Jia J, Yao P, Majumder M, Hatzoglou M, Fox
PL. A stress-responsive RNA switch regulates VEGFA
expression. Nature. 2009;457(7231):915–919.
10. Ambros V. The functions of animal microRNAs.
Nature. 2004;431(7006):350–355.
11. Bartel DP. MicroRNAs: target recognition and
regulatory functions. Cell. 2009;136(2):215–233.
12. Fabian MR, Sonenberg N, Filipowicz W. Regulation
of mRNA translation and stability by microRNAs.
Annu Rev Biochem. 2010;79:351–379.
13. Sasaki T, Shiohama A, Minoshima S, Shimizu N.
Identification of eight members of the Argonaute
family in the human genome small star, filled.
Genomics. 2003;82(3):323–330.
14. Wang B, Li S, Qi HH, Chowdhury D, Shi Y, Novina
CD. Distinct passenger strand and mRNA cleavage
activities of human Argonaute proteins. Nat Struct
Mol Biol. 2009;16(12):1259–1266.
15. Fasanaro P, et al. MicroRNA-210 modulates
endothelial cell response to hypoxia and inhibits
the receptor tyrosine kinase ligand Ephrin-A3.
J Biol Chem. 2008;283(23):15878–15883.
16. Ghosh G, et al. Hypoxia-induced microRNA-424
expression in human endothelial cells regulates
HIF-alpha isoforms and promotes angiogenesis.
J Clin Invest. 2010;120(11):4141–4154.
17. Huang X, et al. Hypoxia-inducible mir-210 regu-
lates normoxic gene expression involved in tumor
initiation. Mol Cell. 2009;35(6):856–867.
18. Kulshreshtha R, et al. A microRNA signature of
hypoxia. Mol Cell Biol. 2007;27(5):1859–1867.
19. Wang S, Olson EN. AngiomiRs--key regula-
tors of angiogenesis. Curr Opin Genet Dev. 2009;
19(3):205–211.
20. John B, Enright AJ, Aravin A, Tuschl T, Sander C,
Marks DS. Human MicroRNA targets. PLoS Biol.
2004;2(11):e363.
21. Krek A, et al. Combinatorial microRNA target pre-
dictions. Nat Genet. 2005;37(5):495–500.
22. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP,
Burge CB. Prediction of mammalian microRNA
targets. Cell. 2003;115(7):787–798.
23. Hutvagner G, Zamore PD. A microRNA in a
multiple-turnover RNAi enzyme complex. Science.
2002;297(5589):2056–2060.
24. Pillai RS, et al. Inhibition of translational initiation
by Let-7 MicroRNA in human cells. Science. 2005;
309(5740):1573–1576.
25. Brown JM, Giaccia AJ. The unique physiology of
solid tumors: opportunities (and problems) for
cancer therapy. Cancer Res. 1998;58(7):1408–1416.
26. Schoch HJ, Fischer S, Marti HH. Hypoxia-induced
vascular endothelial growth factor expression
causes vascular leakage in the brain. Brain. 2002;
125(Pt 11):2549–2557.
27. Connolly E, Braunstein S, Formenti S, Schneider
RJ. Hypoxia inhibits protein synthesis through a
4E-BP1 and elongation factor 2 kinase pathway
controlled by mTOR and uncoupled in breast can-
cer cells. Mol Cell Biol. 2006;26(10):3955–3965.
28. Fahling M. Cellular oxygen sensing, signalling and
how to survive translational arrest in hypoxia. Acta
Physiol (Oxf). 2009;195(2):205–230.
29. Thomas JD, Johannes GJ. Identification of mRNAs
that continue to associate with polysomes during
hypoxia. RNA. 2007;13(7):1116–1131.
30. Lang KJ, Kappel A, Goodall GJ. Hypoxia-inducible
factor-1alpha mRNA contains an internal ribo-
some entry site that allows efficient translation
during normoxia and hypoxia. Mol Biol Cell. 2002;
13(5):1792–1801.
31. Semenza GL. Hypoxia-inducible factor 1: master
regulator of O2 homeostasis. Curr Opin Genet Dev.
1998;8(5):588–594.
32. Kwiecinski M, et al. Expression of platelet-derived
growth factor-C and insulin-like growth factor I
in hepatic stellate cells is inhibited by miR-29. Lab
Invest. 2012;92(7):978–987.
33. Yin KJ, Olsen K, Hamblin M, Zhang J, Schwende-
man SP, Chen YE. Vascular endothelial cell-specific
microRNA-15a inhibits angiogenesis in hindlimb
ischemia. J Biol Chem. 2012;287(32):27055–27064.
34. Carmeliet P, et al. Synergism between vascular
endothelial growth factor and placental growth fac-
tor contributes to angiogenesis and plasma extrav-
asation in pathological conditions. Nat Med. 2001;
7(5):575–583.
35. Green CJ, et al. Placenta growth factor gene expres-
sion is induced by hypoxia in fibroblasts: a central
role for metal transcription factor-1. Cancer Res.
2001;61(6):2696–2703.
36. Kourembanas S, Hannan RL, Faller DV. Oxygen
tension regulates the expression of the platelet-
derived growth factor-B chain gene in human
endothelial cells. J Clin Invest. 1990;86(2):670–674.
37. Semenza GL. Vascular responses to hypoxia and isch-
emia.ArteriosclerThrombVascBiol.2010;30(4):648–652.
38. Wu C, et al. Hypoxia potentiates microRNA-medi-
ated gene silencing through posttranslational
modification of Argonaute2. Mol Cell Biol. 2011;
31(23):4760–4774.
39. Ho JJ, et al. Functional importance of Dicer protein
in the adaptive cellular response to hypoxia. J Biol
Chem. 2012;287(34):29003–29020.
40. Diederichs S, Haber DA. Dual role for argonautes
in microRNA processing and posttranscriptional
regulation of microRNA expression. Cell. 2007;
131(6):1097–1108.
41. Johnson CD, et al. The let-7 microRNA represses
cell proliferation pathways in human cells. Cancer
Res. 2007;67(16):7713–7722.
42. Shell S, et al. Let-7 expression defines two differenti-
ation stages of cancer. Proc Natl Acad Sci U S A. 2007;
104(27):11400–11405.
43. Martello G, et al. A MicroRNA targeting dicer for
metastasis control. Cell. 2010;141(7):1195–1207.
44. Chen HY, et al. miR-103/107 promote metastasis
of colorectal cancer by targeting the metastasis
suppressors DAPK and KLF4. Cancer Res. 2012;
72(14):3631–3641.
45. Tokumaru S, Suzuki M, Yamada H, Nagino M,
Takahashi T. let-7 regulates Dicer expression and
constitutes a negative feedback loop. Carcinogenesis.
2008;29(11):2073–2077.
46. Lu C, Meyers BC, Green PJ. Construction of small
RNA cDNA libraries for deep sequencing. Methods.
2007;43(2):110–117.
47. Kulshreshtha R, Davuluri RV, Calin GA, Ivan M.
A microRNA component of the hypoxic response.
Cell Death Differ. 2008;15(4):667–671.
48. Wingender E, Karas H, Knüppel R. TRANSFAC
database as a bridge between sequence data librar-
ies and biological function. Pac Symp Biocomput.
1997:477–485.
49. Ogel ZB, McPherson MJ. Efficient deletion muta-
genesis by PCR. Protein Eng. 1992;5(5):467–468.
50. Lian SL, Li S, Abadal GX, Pauley BA, Fritzler MJ,
Chan EK. The C-terminal half of human Ago2
binds to multiple GW-rich regions of GW182 and
requires GW182 to mediate silencing. RNA. 2009;
15(5):804–813.
51. Krutzfeldt J, et al. Silencing of microRNAs in vivo
with ‘antagomirs’. Nature. 2005;438(7068):685–689.
52. Passaniti A, et al. A simple, quantitative method for
assessing angiogenesis and antiangiogenic agents
using reconstituted basement membrane, hepa-
rin, and fibroblast growth factor. Lab Invest. 1992;
67(4):519–528.
53. Bonauer A, et al. MicroRNA-92a controls angio-
genesis and functional recovery of ischemic tissues
in mice. Science. 2009;324(5935):1710–1713.