The IRF family of proteins involves in the tumor progression. However, but the functions of IRF5 in the tumorigenesis are largely unknown. Here, IRF5 was found to be up-regulated in hepatocellular carcinoma (HCC). Interfering with IRF5 inhibited the growth and tumorigenic ability of HCC cells.
Applying new techniques to blood assays in cats has enabled researchers to reduce the amount of blood needed nutrition study sample collection studies by 80%, with concomitant benefits for animal welfare. Presented at the Waltham InternationaI Nutrition Science Symposium, October 2016, Chicago
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
Applying new techniques to blood assays in cats has enabled researchers to reduce the amount of blood needed nutrition study sample collection studies by 80%, with concomitant benefits for animal welfare. Presented at the Waltham InternationaI Nutrition Science Symposium, October 2016, Chicago
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Interleukin16 and Interleukin 28B Genes Polymorphism in HBV Infected Saudi pa...iosrjce
The course of hepatitis B virus (HBV) infection is variable depending on many factors. In this study,
we investigated single nucleotide polymorphisms of interleukin-28B and interleukin-16 as possible host factors
which may determine the occurrence of hepatocellular carcinoma (HCC) in Saudi patients. Chronic hepatitis B
(CHB) patients (75), HCC patients (42), and healthy controls (70) were analyzed for polymorphisms of the IL16
and IL28B genes using PCR and restriction fragment length polymorphism (RFLP). Results showed that HCC
and chronic HBV patients had higher prevalence of rs11556218TG genotype than controls. The rs11556218GG
genotype was higher among HCC (14.4%) compared to chronic HBV (2.7%) patients. The IL-16 genotype
rs4072111CT was higher among HCC (47.6%) and chronic HBV (46.7%) patients than controls (28.6%). The
rs4072111TT genotype was higher among HCC patients compared to the other two groups. The T allele
frequency was higher among HCC patients than controls. The CT and TT of the IL-28B rs12979860 genotype
were significantly less frequent in chronic HBV and HCC patients. The IL-28B rs8099917 TG genotype was
more frequent among HCC (19%) compared to chronic HBV (8%) patients. However, no significant difference
was detected in the allele distribution.
Lipopolysaccharide promotes the metastatic Potential of Lung Carcinoma Cells ...ijtsrd
Lipopolysaccharide (LPS) stimulated TLR-4 mediated signalling has been shown to accentuate the metastasis in a variety of cancers. In a murine mammary cancer model, LPS has been found to trigger lung metastasis. However there are very few studies which report the pro-tumor properties of LPS in lung cancer. In this study we investigated the effect of LPS on Fibronectin induced metastatic progression in human lung cancer cells. Lower concentrations of LPS exhibited a pro-proliferative effect on A549 cells. Using such concentrations of LPS to stimulate A549 cells, expression of Fibronectin mRNA increased in comparison to control. 0.5 -µg of LPS treatment led to the two fold increase in mRNA expression of Fibronectin. Moreover, LPS stimulation augmented the migratory ability of A549 cells as assessed by wound healing assay. This increase in migration of A549 cells was in agreement with the expression pattern of Fibronectin induced by LPS stimulation. Taken together this study presents a preliminary account of the role of LPS in inducing the expression of Fibronectin and metastatic progression in lung carcinoma. Asif Amin | Taseem A. Mokhdomi | Asrar H. Wafai | Raies A. Qadri"Lipopolysaccharide promotes the metastatic Potential of Lung Carcinoma Cells by Upregulating the Expression of Fibronectin" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-1 , December 2017, URL: http://www.ijtsrd.com/papers/ijtsrd7164.pdf http://www.ijtsrd.com/biological-science/cell-biology/7164/lipopolysaccharide-promotes-the-metastatic-potential-of-lung-carcinoma-cells-by-upregulating-the-expression-of-fibronectin/asif-amin
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Anemia is a common condition of cancer patients. This is because cancers cause inflammation that decrease red blood cell production. In addition, many chemotherapies are myelosuppressive, meaning they slow down the production of new blood cells by the bone marrow.
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Cancer is a prime public health burden that accounts for approximately 9.9 million deaths worldwide. Despite recent advances in treatment regimen and huge capital investment in the pharmaceutical sector, there has been little success in improving the chances of survival of cancer patients.
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Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Interleukin16 and Interleukin 28B Genes Polymorphism in HBV Infected Saudi pa...iosrjce
The course of hepatitis B virus (HBV) infection is variable depending on many factors. In this study,
we investigated single nucleotide polymorphisms of interleukin-28B and interleukin-16 as possible host factors
which may determine the occurrence of hepatocellular carcinoma (HCC) in Saudi patients. Chronic hepatitis B
(CHB) patients (75), HCC patients (42), and healthy controls (70) were analyzed for polymorphisms of the IL16
and IL28B genes using PCR and restriction fragment length polymorphism (RFLP). Results showed that HCC
and chronic HBV patients had higher prevalence of rs11556218TG genotype than controls. The rs11556218GG
genotype was higher among HCC (14.4%) compared to chronic HBV (2.7%) patients. The IL-16 genotype
rs4072111CT was higher among HCC (47.6%) and chronic HBV (46.7%) patients than controls (28.6%). The
rs4072111TT genotype was higher among HCC patients compared to the other two groups. The T allele
frequency was higher among HCC patients than controls. The CT and TT of the IL-28B rs12979860 genotype
were significantly less frequent in chronic HBV and HCC patients. The IL-28B rs8099917 TG genotype was
more frequent among HCC (19%) compared to chronic HBV (8%) patients. However, no significant difference
was detected in the allele distribution.
Lipopolysaccharide promotes the metastatic Potential of Lung Carcinoma Cells ...ijtsrd
Lipopolysaccharide (LPS) stimulated TLR-4 mediated signalling has been shown to accentuate the metastasis in a variety of cancers. In a murine mammary cancer model, LPS has been found to trigger lung metastasis. However there are very few studies which report the pro-tumor properties of LPS in lung cancer. In this study we investigated the effect of LPS on Fibronectin induced metastatic progression in human lung cancer cells. Lower concentrations of LPS exhibited a pro-proliferative effect on A549 cells. Using such concentrations of LPS to stimulate A549 cells, expression of Fibronectin mRNA increased in comparison to control. 0.5 -µg of LPS treatment led to the two fold increase in mRNA expression of Fibronectin. Moreover, LPS stimulation augmented the migratory ability of A549 cells as assessed by wound healing assay. This increase in migration of A549 cells was in agreement with the expression pattern of Fibronectin induced by LPS stimulation. Taken together this study presents a preliminary account of the role of LPS in inducing the expression of Fibronectin and metastatic progression in lung carcinoma. Asif Amin | Taseem A. Mokhdomi | Asrar H. Wafai | Raies A. Qadri"Lipopolysaccharide promotes the metastatic Potential of Lung Carcinoma Cells by Upregulating the Expression of Fibronectin" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-1 , December 2017, URL: http://www.ijtsrd.com/papers/ijtsrd7164.pdf http://www.ijtsrd.com/biological-science/cell-biology/7164/lipopolysaccharide-promotes-the-metastatic-potential-of-lung-carcinoma-cells-by-upregulating-the-expression-of-fibronectin/asif-amin
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Anemia is a common condition of cancer patients. This is because cancers cause inflammation that decrease red blood cell production. In addition, many chemotherapies are myelosuppressive, meaning they slow down the production of new blood cells by the bone marrow.
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Fibrous
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Adipose (Fatty)
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15 projections are taken with each combo exposure (7.5) (-7.5)
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Skeletal muscle channelopathy are rare heterogeneous episodic disorders with marked genotypic and phenotypic variability resulting in periodic paralysis, and falls in young people which often misdiagnosed or undiagnosed due to its rarity, often the symptoms are miscommunicated to the treating phycision due to its episodic nature and not uncommonly physical examination by the time patient attend the clinic or hospital will be unremarkable apart from periodic muscle paralysis where patient will presented to ED with flaccid weakness,
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Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
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These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
2. clinicsofoncology.com 2
Volume 6 Issue 7 -2022 Research Article
PKM2 to inhibit the glycolysis and tumorigenic ability of HCC
cells [23,24]. The expression of TRIM35 and PKM2 in HCC pre-
dicts the prognosis for HCC patients [23,24]. To further elucidat-
ing the functions of TRIM35 in HCC, the proteins interacting with
TRIM35 were predicted using the STRING database. IRF5 is one
of the potential proteins to interact with TRIM35. In the present
work, the abundance of IRF5 in HCC was studied. In addition, the
role of IRF5 in HCC cells and the regulating effect of TRIM35 on
IRF5 were also examined.
3. Materials and Methods
3.1. Cell culture
The HepG2, Huh7 and QGY cells were obtained from the Cell
Bank, Chinese Academy of Sciences. The cells were cultured in
DMEM medium with 10% fetal bovine serum (FBS) and antibiot-
ics (100 U/mL of penicillin and 100 µg/ml of streptomycin). Cells
were in a constant temperature incubator (5% CO2
, 37℃). Lipo-
fectamine 8000 was used for the cell transfection.
3.2. Clinical samples
The clinical samples were collected from the Shanghai Eastern
Hepatobiliary Surgery Hospital after the study was approved by
their Ethics Committee. All of the patients have signed the consent
form.
3.3. Immunohistochemistry (IHC)
The array of HCC tissues was provided by the Shanghai Eastern
Hepatobiliary Surgery Hospital. Dewaxing, rehydration and anti-
gen recovery in the 100°C EDTA solution were done. Then, the
activity of endogenous peroxidase was blocked. Before being in-
cubated with the IRF5 antibody (Sigma, HPA046700, 1:100) at
4°C overnight, the tissue sections were washed with PBS. On the
next day, before being incubated with secondary antibody at room
temperature for 1h, the tissue sections were washed with PBS. The
signal was developed using 3,3,0-diaminobenzidine (DAB), and
the nucleus were stained using hematoxylin. Both the staining in-
tensity and protein expression level were automatically scored by
the Vectra 2.0 system.
3.4. qPCR
RNA was extracted with TRIzol (Invitrogen) and reversely tran-
scribed into cDNA using the PrimeScript™ RT kit (Takara) ac-
cording to the instructions. qPCR was performed using The SYBR
Green kit and CFX96 real-time fluorescent quantitative PCR de-
tection system (Bio-Rad, Richmond, CA, USA). The levels of the
transcripts were calculate using the 2−ΔΔ
Ct method. The primer
sequences for IRF5 were: Forward primer, 5’-tgtgcccttaacaagag-
ccg-3’; Reverse primer, 5’-ctctgtgaggctcaggcttg-3’.
3.5. Western blot
The cells were cleaned twice with PBS, and lysed on ice with the
RIPA Lysis buffer, which contains the protease inhibitor and phos-
phatase inhibitor. The supernatant was collected after the cell ly-
sis was centrifuged, and the protein concentration was quantified
using the BCA protein detection kit. An equal amount of protein
was taken for the SDS-PAGE analysis. After separation, the pro-
tein was transferred onto the PVDF membrane and incubated with
a specific primary antibody at 4°C overnight. Then, the membrane
was incubated with the HRP-conjugated secondary antibody for
1-2h. The immune signal was detected with a chemiluminescence
reagent (Milliwell, WBKLS0050), and analyzed with Image Lab
software. The primary antibodies used in this experiment were as
follows: IRF5 (Sigma, HPA046700, 1:100), tubulin (Santa Cruz
Biotechnolog, sc-5286, 1:4000), Flag (Sigma, F9291; 1:3,000),
HA(Sigma, H3663, 1:2000), TRIM35 (Sigma, HPA019647,
1:100), Ubiquitin (proteintech, 10201-2-AP, 1:1000).
3.6. CCK8
Cells were seeded into a 96-well plate, and each well contained
1000 cells. 18 hours later, cells were incubated with a fresh me-
dium containing 10% CCK8 for 2 hours. Then, OD450 nm was
evaluated. The measurement was performed on day 1, 3, 5 and 7,
respectively.
3.7. Edu assay
Cells were seeded into a 96-well plate, and each well contained
1000 cells. 18 hours later, the proliferation was evaluated with
the Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, C10310-1).
Photos were taken by the fluorescent microscope for analysis.
3.8. Soft agar assay
When the confluence reached 60-70%, the cells were digested, and
a cell suspension was prepared. Lower-layer gel (20% FBS, 40%
2×RPMI1640 (Basal Medium Eagle) 40% 1.25% Agar) was pre-
pared. 400μL of gel was added to each well in the 24-well plate.
The 24-well plate with the gel was placed in an incubator at 37°C.
The gel was solidified for later use. Upper-layer gel (25% FBS,
37.5% 2×RPMI1640, 37.5% 1% Agar, 0.8% 2mM L-glutamine)
was prepared and mixed evenly with the cell suspension. 400μl
(containing 1000 cells) was added to each well and placed in a
constant temperature incubator (37°C, 5% CO2
) for two weeks. 5
fields were selected randomly under the microscope for colonies
counting.
3.9. Immunoprecipitation
In order to detect the interaction between exogenous IRF5 (Flag-
IRF5) and TRIM35 (HA-TRIM35), the Flag-IRF5 and HA-
TRIM35 plasmids were transferred into 293T. 48h after transfec-
tion, the cells were lysed with an IP lysis buffer (50mM Tris-HCl,
pH 8.0, 150mM NaCl, 1 %NP-40, protease and phosphatase in-
hibitor), with the supernatant collected. The beads coupling an-
ti-Flag antibody (Sigma, A2220) were added to the supernatant
for incubation overnight at 4°C. The next day, the beads were
washed 3 times in wash buffer (50mM Tris-HCl (pH 8.0), 150mM
NaCl, 1%NP-40), with 1×loading buffer added, heated at 100°C
for 5min, and then the supernatant was taken for western blot
3. clinicsofoncology.com 3
Volume 6 Issue 7 -2022 Research Article
analysis. To detect whether there was any interaction between the
endogenously expressed IRF5 and TRIM35 in HCC cells, an IP
lysis buffer containing protease and a phosphatase inhibitor was
sued for lysis. An equal amount of protein was taken, and 0.25μg
of IRF5 antibody was added for incubation overnight at 4°C. The
next day, 40μL of Protein A/G beads (bimake, B23202) was added
for another incubation overnight at 4°C. The beads were washed
for 3 times with the wash buffer, and then 1×loading buffer was
added for western blotting analysis.
3.10. Data statistics
Data was expressed as mean ± SD. The data were analyzed using
the t test. A survival curve was plotted by the Kaplan-Meier meth-
od, while the log-rank test was used for analysis. GraphPad Prism
8 and SPSS 17.0 were used for statistical analysis.
3. Results
3.1. IRF5 is overexpressed in HCC
To study the abundance of IRF5, the transcripts of IRF5 in HCC
tissues and matched adjacent non-cancerous tissues were mea-
sured. It was found that the levels of IRF5 transcripts in the cancer
tissues were higher than that in normal tissues (Figure 1A). When
the levels of IRF5 mRNA in the tumors was compared with that of
IRF5 in the paired adjacent tissues, higher levels of IRF5 mRNAin
the tumor tissues were observed (Figure 1B). After this, the protein
levels of IRF5 were measured. In the tested HCC tissues, the level
of IRF5 protein was relatively high (Figure 1C). Moreover, the
results were verified using immunohistochemistry staining (Fig-
ure 1D). In the Human Protein Atlas database, IRF5 expression is
inversely correlated with the outcome of HCC (Figure 1E). These
observations indicate that IRF5 might be important for the HCC
development.
Figure 1: IRF5 expression is upregulated in hepatocellular carcinoma.
(A) The levels of IRF5 mRNA in 30 cancer samples (cancer) and 30 adjacent tissues (normal) were examined by qPCR. 18S, internal control. The Ct values
(18S-IRF5) were plotted on the ordinate and analyzed. (B) The mRNA levels of IRF5 in paired cancer tissues (cancer) and non-cancerous tissues (normal)
were analyzed. (C) The levels of IRF5 protein in 5 cancerous tissues (C) and paired non-cancerous tissues (N) were examined by Western blotting. (D) The
levels of IRF5 protein in the tumor samples and adjacent non-cancerous tissue was examined by IHC. (E) Mining the HPA database and analyzing the cor-
relation between IRF5 transcripts and the HCC outcome.
4. clinicsofoncology.com 4
Volume 6 Issue 7 -2022 Research Article
3.2. IRF5 promotes the growth and colony formation of HCC
cells
To study the IRF5’s functions, exogenous IRF5 was forced ex-
pressed in Huh7 and HepG2 cells, allowing the functions of IRF5
in cell growth to be detected (Figure 2A). In the CCK8 assay, the
promotion of the growth was observed upon the overexpression of
IRF5 in the HCC cells (Figure 2B). After this, the anchorage-in-
dependent growth of HCC cells was evaluated. It shows that the
expression of IRF5 promoted the anchorage-independent growth
(Figure 2C-D). IRF5 in Huh7 and QGY cells was then interfered
with two shRNA sequences (Figure 3A). It was found that the
growth of HCC cells in the liquid medium were inhibited after the
expression of IRF5 was down-regulated (Figure 3B). Moreover,
down-regulation of IRF5 impaired the growth of HCC cells on the
soft agar (Figure 3C-D). Cell growth is the result of apoptosis and
proliferation. The effect of the expression of IRF5 on the prolifer-
ation was detected by means of an EdU assay. The results showed
that silencing IRF5 impaired the proliferation of HCC cells (Fig-
ure 3E-F).
Figure 2: IRF5 promotes the growth of hepatocellular carcinoma cells.
(A) The exogenous Flag-IRF5 was forced expressed in Huh7 and HepG2 cells, and examined using western blot. (B) Cell growth was evaluated with
CCK8. (C-D) The anchorage-independent growth of HCC cells was examined after the overexpression of IRF5. The colonies were counted and statis-
tically analyzed.
*, P<0.05; **, P<0.01.
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Volume 6 Issue 7 -2022 Research Article
Figure 3: Silencing IRF5 inhibits the proliferation.
(A) The IRF5 knockdown efficiency was examined with Western blot. (B) Cell growth was analyzed by CCK8 assay. (C-D) The anchorage-independent
growth of HCC cells was examined after the knockdown of IRF5. The colonies were counted and statistically analyzed. (E-F) Cell proliferation was
analyzed with EdU assay. EdU positively stained cells were counted and statistically analyzed. *, P<0.05; **, P<0.01.
3.3. TRIM35 regulates the stability of the IRF5 protein
In order to study the regulation of IRF5 in HCC, the proteins in-
teracting with IRF5 were searched with the help of STRING da-
tabase. The results showed that IRF5 interacted with the E3 ubiq-
uitin ligase TRIM35 (Figure 4A). The binding between IRF5 and
TRIM35 was evaluated by means of a co-immunoprecipitation
assay (CO-IP). In Huh7 and HepG2 cells, the exogenously ex-
pressed HA-TRIM35 interacted with the exogenously expressed
Flag-IRF5 (Figure 4B). The CO-IP results also showed that the
endogenously expressed IRF5 and TRIM35 in Huh7 cells had
formed a complex (Figure 4C). Moreover, TRIM35 promoted the
ubiquitination and degradation of IRF5 (Figure 4D).
Figure 4: TRIM35 promotes the degradation of IRF5 in HCC cells.
(A) Mining the STRING database for the binding proteins of TRIM35. (B) The binding of ectopically expressed HA-TRIM35 and Flag-IRF5 was
examined using immunoprecipitation assay in Huh7 cells. (C) The binding of endogenously expressed TRIM35 and IRF5 was examined using immu-
noprecipitation assay in Huh7 cells. (D) The levels of the ubiquitinated IRF5 by TRIM35 were elevated.
6. clinicsofoncology.com 6
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3.4. TRIM35 is down-regulated in HCC and negatively cor-
related with the expression of IRF5
The expression of TRIM35 in HCC was analyzed using a publicly
available database. The cBioportal database shows that TRIM35
is deleted in HCC (Figure 5A). The HPA database shows that the
expression of TRIM35 is positively correlated with the HCC out-
come (Figure 5B). The levels of TRIM35 mRNA remained lower
in cancerous tissues (Figure 5C). The level of IRF5 protein and
TRIM35 protein in 20 cases of HCC was then measured by means
of immunocytochemistry (IHC). The negative correlation between
their expression was observed (Figure 5D). The level of IRF5 pro-
tein was high in the HCC tissue with low-expressed TRIM35 (Fig-
ure 5D).
Figure 5: The expression of TRIM35 and IRF5 was negatively correlated in HCC.
(A) Mining the cBioportal database for the mutation, amplification and deletion of TRIM35 in HCC. (B) Mining the HPA database to analyze the cor-
relation between TRIM35 mRNA level and survival. (C) The levels of TRIM35 mRNA were evaluated by qPCR. 18S, internal control. The Ct values
(18S-IRF5) were plotted on the ordinate and analyzed. (D) The correlation between the expression of IRF5 and TRIM35 in the clinical HCC tissues
was analyzed. The protein levels of IRF5 and TRIM35 in the 20 HCC tissues were examined using IHC and scored.
4. Discussion
It has been reported that IRF5 inhibits the replication of HCV,
suggesting that it may also inhibit HCV-related HCC progression
[19]. But in the HPA database, IRF5 is inversely correlated with
the outcome of HCC patients. Therefore, further evidence is need-
ed to clearly support the oncogenic roles of IRF5 in HCC. It was
seen that the levels of IRF5 mRNA and protein were up-regulat-
ed in HCC, and that IRF5 expression accelerated the growth and
colony formation of HCC cells. TRIM35 was low-expressed in
the HCC specimen, and it promoted the degradation of IRF5 by
binding to IRF5; in the clinical tissues, IRF5 expression was re-
versely correlated with TRIM35 expression. These data indicate
that IRF5 is essential for the HCC progression. The most import-
ant finding from this study is that IRF5 promotes cell growth. The
IRF family of proteins, such as IRF1 and IRF2, have been proven
to have completely opposite effects on the growth of ESCC [16].
IRF8 promotes the proliferation of acute myelocytic leukemia
(AML) cells [25]. This shows that the IRF protein family have a
regulating effect on the growth of tumor cells. In addition, the IRF
family interferes with tumor progression by reshaping the tumor
immunity microenvironment. For example, the GM/CSF/IRF5
pathway enhances antitumor immunity by activating the Type 1
T-cell response [26]. This indicates that IRF proteins can regulate
tumorigenesis via different mechanisms. This study reveals the
ubiquitination of IRF5 by TRIM35. Up to date, IRF5 is rarely to
be post-transcriptionally modified. IRF5 can be acetylated by his-
tone acetyltransferase (HAT)[27]. The ubiquitination of IRF5 can
be catalyzed by pellino-1, which helps M1 macrophages regulate
obesity [28]. The results of this study show that TRIM35 mediates
the degradation of IRF5. Coincidentally, many of the existing stud-
ies show that TRIM35 can also regulate the interferon signaling
pathways. The findings of this study further support the previous
7. clinicsofoncology.com 7
Volume 6 Issue 7 -2022 Research Article
conclusions. In conclusion, this work reveals the promoting func-
tion of IRF5 in hepatocarcinogenesis, and the regulation of IRF5
by TRIM35, suggesting IRF5 as a promising targe.
5. Acknowledgement
Not applicable.
6. Disclosure of Conflict of Interests
No potential conflict of interest was reported by the author(s).
7. Funding
This work was supported by foundation of Shanghai Institute of
Rehabilitation with Integrated Western and Chinese Traditional
Medicine (082)
8. Data Availability
The data supporting the results reported in the manuscript can be
acquired by the corresponding author.
9. Ethics Statement
All patients participated in this study have signed informed con-
sent, and this research was approved by the Ethics Committee of
Shanghai Eastern Hepatobiliary Surgery Hospital.
10. Author Contribution
Fang Ying and Lu Zhihui collected the clinical tissues, performed
the IHC staining, and studied the molecular mechanisms; Liu
Bangzhong performed the CCK8 assay; Yang Mingzhen per-
formed the soft agar assay; Li Nan provided the clinical tissues;
Wang Ping conceived and designed the experiments, analyzed the
data, reviewed drafts of the paper, and approved the final draft.
11. Consent for Publication
All authors agreed to the publication of the manuscript.
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