REVIEWSMicroRNAs in body fluids—the mixof hormones and biomarkersMaria Angelica Cortez, Carlos Bueso-Ramos, Jana Ferdin, G...
REVIEWS Key points ■ A single microRNA (miRNA) can target and regulate hundreds or thousands                              ...
REVIEWStissues.38 MiRNA-expression signatures also correlate                                                              ...
REVIEWS                    bladder and esophageal cancer risk.58,59 In breast cancer,               cancer (Table 1). Conf...
REVIEWS Table 1 | A compendium of circulating miRNAs with potential as biomarkers for cancer miRNAs                       ...
REVIEWS                    malignancies. For instance, in a study in acute leukemias,             multivesicular bodies (M...
REVIEWSnecessary to unveil how miRNAs are sorted into exo-                                                                ...
REVIEWS Box 1 | MiRNAs—the ‘oldest’ hormones                                                               and differences...
REVIEWScircumventing unnecessary treatments and collateral side                  RB1, TP53, and PTEN), and chromosomal tra...
REVIEWS34. Ciafre, S. A. et al. Extensive modulation of a set            the AML1/ETO oncoprotein. Cancer Cell 12,        ...
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A descoberta da capacidade de certos RNAs serem capazes de bloquear o RNA mensageiro (por serem complementares base a base) há alguns anos,abriu as portas do tratamento de inibição da expressão nucleica de proteínas anormais, responsáveis por doenças. Agora, surgem os miRNA que são produzidos em introns ou exons de gens formadores de proteínas. Estão relacionados ao câncer e diabetes.

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  1. 1. REVIEWSMicroRNAs in body fluids—the mixof hormones and biomarkersMaria Angelica Cortez, Carlos Bueso-Ramos, Jana Ferdin, Gabriel Lopez-Berestein, Anil K. Soodand George A. CalinAbstract | Since the discovery of microRNAs (miRNAs), the study of these small noncoding RNAs has steadilyincreased and more than 10,000 papers have already been published. The great interest in miRNAs reflectstheir central role in gene-expression regulation and the implication of miRNA-specific aberrant expression inthe pathogenesis of cancer, cardiac, immune-related and other diseases. Another avenue of current researchis the study of circulating miRNAs in serum, plasma, and other body fluids—miRNAs may act not only withincells, but also at other sites within the body. The presence of miRNAs in body fluids may represent a gold mineof noninvasive biomarkers in cancer. Since deregulated miRNA expression is an early event in tumorigenesis,measuring circulating miRNA levels may also be useful for early cancer detection, which can contribute greatlyto the success of treatment. In this Review, we discuss the role of fluid-expressed miRNAs as reliable cancerbiomarkers and treatment-response predictors as well as potential new patient selection criteria for clinicaltrials. In addition, we explore the concept that miRNAs could function as hormones.Cortez, M. A. et al. Nat. Rev. Clin. Oncol. 8, 467–477 (2011); published online 7 June 2011; doi:10.1038/nrclinonc.2011.76 until 2000, when another small RNA, let‑7, was identified Continuing Medical Education online and found to be conserved in many species,3,4 that a new This activity has been planned and implemented in accordance layer of complexity in the regulation of gene expression with the Essential Areas and policies of the Accreditation Council was unveiled. The discovery of the post-transcriptional for Continuing Medical Education through the joint sponsorship of Medscape, LLC and Nature Publishing Group. Medscape, LLC is silencing of target mRNAs5 by small RNAs was a revolu- accredited by the ACCME to provide continuing medical education tionary step in the understanding of genetic-information for physicians. control. Further studies provided evidence that these Medscape, LLC designates this Journal-based CME activity for microRNAs (miRNAs) are members of a large class of a maximum of 1 AMA PRA Category 1 Credit(s)TM. Physicians non-coding RNAs of approximately 22 nucleotides in should claim only the credit commensurate with the extent of their participation in the activity. length, which regulate most genes in the human genome.6 All other clinicians completing this activity will be issued a MiRNAs are strongly conserved between vertebrates, certificate of participation. To participate in this journal CME invertebrates, and plants,7 and are transcribed from indivi- activity: (1) review the learning objectives and author disclosures; dual genes sometimes clustered and located intergenic or (2) study the education content; (3) take the post-test and/or in introns or exons of protein-coding genes.8 complete the evaluation at nrclinonc; (4) view/print certificate. MiRNA biogenesis involves the maturation of miRNA Released: 7 June 2011; Expires: 7 June 2012 precursors, assembly of the mature miRNA into micro- processor complexes and the regulation of gene expres- Learning objectives sion of protein-coding genes by degrading or blocking Upon completion of this activity, participants should be able to: translation of mRNA targets (Figure 1).9 First, miRNA is 1. Analyze the relationship between microRNAs and cancer 2. Distinguish specific microRNAs associated with cancer transcribed in the nucleus by RNA polymerase II as long outcomes and capped precursor primary miRNA (pri-miRNA).10,11 3. Evaluate the presence of microRNAs in body fluids The next step is the production of a precursor miRNA 4. Describe the measurement of microRNAs in body fluids (pre-miRNA) by the ribonuclease (RNase) III Drosha enzyme and the processing of the double-stranded DNA- The University of Texas MD Anderson CancerIntroduction binding protein DGCR8.12 Pre-miRNAs are actively Center, 1515 HolcombeIn 1993, Victor Ambros’1 and Gary Ruvkun’s2 groups exported to the cytoplasm by the nuclear export receptor Boulevard, Houston, TX 77030, USAdiscovered that the abundance of the protein LIN14 in exportin 5 and then processed by the RNase III endo- (M. A. Cortez,Caenorhabditis elegans was regulated by a small RNA nuclease Dicer protein along with the double-stranded C. Bueso‑Ramos,product encoded by the lin‑4 gene. However, it was not transactivation-responsive RNA-binding protein (TRBP), J. Ferdin, G. Lopez‑Berestein, resulting in a small double-strand RNA structure of about A. K. Sood, G. A. Calin). 22 nucleotides.13,14 This miRNA duplex is unwound intoCompeting interests Correspondence to:The authors, the journal Chief Editor L. Hutchinson and the CME the mature single-strand form and incorporated into the G. A. Calinquestions author C. P. Vega declare no competing interests. RNA-induced silencing complex (RISC), which guides gcalin@mdanderson.orgNATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 8 | AUGUST 2011 | 467 © 2011 Macmillan Publishers Limited. All rights reserved
  2. 2. REVIEWS Key points ■ A single microRNA (miRNA) can target and regulate hundreds or thousands POL II of mRNAs; aberrant miRNA expression is involved in the initiation of many Promotor miRNA diseases, including cancer ■ MiRNAs are potentially useful as biomarkers in cancer diagnosis, prognosis and response to treatment owing to the unique expression profile of each tumor and limited complex transcriptional and translational modifications DGC Drosha R8 ■ The discovery of miRNAs in body fluids opens up the possibility of using them Pri-miRNA as non-invasive biomarkers in cancer detection and as predictors of therapy Pre-miRNA response in clinical trials Frameshift mutation ■ Standardized methods with well-established parameters for miRNA detection Nucleus RAN are necessary to indicate cancer stage, response to treatment, outcome and -GTP Cytoplasm cancer recurrence Exportin 5 Frameshift mutation the complex into the complementary 3 or 5-untranslated TARB GE P M Dicer1 2 GE IN3 region (UTR) of the target mRNA,15–18 open reading Ago2 MIN4 frames, and promoter regions. 19 Variations in the Pre-miRNA expression of Dicer and Drosha mRNA and protein are Post-transcriptional RISC extensive in human cancers,20 and frameshift functional regulation mutations in the biogenesis machinery including XPO5 dsRNA AA AA and TARBP2 occur in human tumors with microsatellite AA instability (Figure 1).21,22 Non-perfect Translational repression Although negative regulation of gene expression occurs via mRNA cleavage or translational repression,23,24 studies Perfect mRNA cleavage have shown that miRNAs can upregulate the expression of their target genes25 and that a single gene can be tar- Figure 1 | MiRNA biogenesis in the cell. MiRNAs are geted by multiple miRNAs.26 In mammals, gene regula- transcribed in the nucleus as pri-miRNA and then processed tion mediated by miRNAs is accomplished by imperfect by Drosha into pre-miRNA. Pre-miRNA molecules are base pairing together with protein translational repression transported from the nucleus to the cytoplasm by exportin 5. of the target gene.27,28 Upon entering the cytoplasm, they are recognized by Dicer. Dicer modulates pre-miRNA and generates dsRNA, which are MiRNAs are involved in virtually all biologic pro- recognized by the RISC complex and converted into single- cesses and, because a single miRNA can target hundreds strand mature miRNA molecules. The RISC complex carries of mRNAs, aberrant miRNA expression is involved in the mature miRNA molecule to complementary miRNA target the initiation of many diseases, including cancer. In this sites within the mRNA molecule, where it affects gene Review, we focus on some paradigms of miRNA involve- expression by miRNA:mRNA sequence complementarity. ment in cancer, on the potential detection of tumor- A consequence of perfect complementarity between specific miRNAs in body fluids and their applicability miRNA:mRNA molecules is mRNA cleavage and degradation. as diagnostic and prognostic markers in cancer. We also Imperfect alignment represses gene translation. Mutations in RNA processing are indicated with red lightning bolts. discuss the concept that miRNAs can act as hormones A frameshift mutation in exportin 5 caused premature codon through their secretion in plasma and address the effects termination and trapped pre-miRNAs in the nucleus.21 Other of their delivery to distant sites in the body. frameshift mutations in the RISC-loading complex subunit TARBP2 causes a loss of function of TARBP2, a secondary Paradigms of miRNAs in cancer defect of Dicer activity and the loss of miRNA machinery MiRNAs are altered in every type of cancer regulation during tumorigenesis.22 Abbreviations: Ago2, Genome-wide miRNA-expression-profiling studies using Argonaute2; dsRNA, double-strand miRNA; miRNA, microRNA; POL II, RNA polymerase II; pre-miRNA, precursor high-throughput technologies have demonstrated that miRNA; pri-miRNA, precursor primary miRNA; RISC, almost all cancer types present a specific profile of upregu- RNA-induced silencing complex. lated and downregulated miRNAs.29,30 Therefore, owing to the unique miRNA-expression profile for each tumor and the lack of complex transcriptional and translational between colonic adenocarcinoma and normal mucosa modifications compared to mRNAs and proteins, the use and also found that levels of miR‑143 and miR‑145 were of miRNAs as biomarkers for cancer has great potential. significantly lower in tumors than in normal tissue.32 For example, by analyzing the chromosome region 13q14, Similarly, other studies found specific signatures of a region that is deleted in more than half of all patients miRNA expression in breast carcinoma,33 primary glio- with B-cell chronic lymphocytic leukemia (B-CLL), it blastoma,34 hepatocellular carcinoma,35 papillary thyroid was demonstrated that miR‑15a and miR‑16a were either carcinoma,36 and lung cancer.37 In a large profiling analysis absent or downregulated in approximately 68% of patients of 540 samples of six solid tumors (lung, breast, stomach, with B-CLL.31 One of the first studies of miRNA in solid prostate, colon, and pancreas), a group of 43 miRNAs was tumors identified 28 miRNAs differentially expressed found to be deregulated compared to matched normal468 | AUGUST 2011 | VOLUME 8 © 2011 Macmillan Publishers Limited. All rights reserved
  3. 3. REVIEWStissues.38 MiRNA-expression signatures also correlate Apoptosis inhibitionwith tumor classification and have proven useful in deter- (p53 pathway) As tumor-suppressor genemining the primary site of cancers of unknown origin.29Metastatic cancers of unknown primary origin were clas- Invasion Cell-cycle miR-125b progressionsified with >90% accuracy based on an expression profileof 48 miRNAs.39 Furthermore, the global-expression miR-125b Cell-cyclepattern of miRNAs distinguished between long-term miR-125b progressionand short-term survivors of pancreatic cancer and also miR-125b Cell proliferationdifferentiated ductal adenocarcinomas of the pancreas As oncogenefrom normal pancreas and chronic pancreatitis with 95% Ovarian canceraccuracy.40 Following the discovery of different expression Neuroblastoma Thyroid cancer Prostate cancer Oral squamous-levels of miRNAs between normal and cancerous tissues, Glioblastoma cell carcinomathe next step has been to attempt to ascertain the impact Thyroid cancer Breastof these small RNAs on tumorigenesis. Figure 2 | A miRNA can function dually as both an oncogene and tumor-suppressorAngels and devils gene depending on the cancer type and cellular context. A duality of function inGrowing evidence has demonstrated that miRNAs can distinct types of cancer has been found for many miRNAs. An example isact as either oncogenes or tumor-suppressor genes. For miR-125b, which has opposite roles (oncogene and tumor suppressor) in differentinstance, miR‑21 is upregulated in various solid tumors cancer types or cell lines. As a tumor suppressor, miR-125b is downregulated inas well as hematologic malignancies, and this potentially ovarian, thyroid, breast, and oral squamous-cell carcinomas, which promotes celloncogenic miRNA regulates important tumor-suppressor proliferation and cell-cycle progression.47,48 On the other hand, miR-125b is an oncogene in cancers such as prostate, thyroid, glioblastoma, and neuroblastoma.genes such as PTEN 41 and PDCD4. 42 Another well- In neuroblastoma cells, miR-125b inhibits apoptosis in a p53-dependent manner,49characterized oncogenic miRNA is miR‑10b that pro- and promotes cell proliferation and invasion in prostate cancer cells.50motes metastasis by suppressing HOXD10, which isthe negative regulator of a gene associated with tumorcell proliferation and metastasis.43 In addition to single MiRNAs and cancer predispositionmiRNAs, clusters of miRNAs such as the miR‑17‑92 Unlike the aberrant miRNA expression in somatic cellscluster also promote proliferation, increase angiogenesis, that can promote tumorigenesis, altered expressionand sustain cancer cell survival via post-transcriptional of miRNA in germline cells may predispose to cancerrepression of target mRNAs.44 development. An explanation for this well-characterized There are also miRNAs that regulate oncogenes and difference in miRNA expression in cancer comparedthus act as tumor suppressors. For example, members with normal cells is that these small RNAs are frequentlyof the tumor-suppressor miRNA let‑7 family are down- located in cancer-associated genomic regions (CAGRs)regulated in many malignancies and inhibit cancer and are often subject to rearrangements, breakpoints,growth by targeting various oncogenes and key regula- loss of heterozygosity, and deletions.52 A paradigm fortors of mitogenic pathways, such as RAS and HMGA2.45 this model is B-CLL, in which miR‑15a and miR‑16‑1 areOther well-established tumor suppressors are the effectors located in the most frequently deleted genomic region,of TP53 activation in the miR‑34 family.46 are downregulated in the majority of cases, and harbor Nonetheless, the initial categorization of miRNAs as germline mutations in familial cases of CLL and breastoncogenes or tumor-suppressor genes based on their cancer. 31 Another example is the miR‑17‑92 cluster,levels of expression in tumors versus normal tissues has which is located in intron 3 of the C13orf25 gene onproven to be inaccurate, as experiments have shown that 13q31.3, a chromosomal region amplified in many typesmany function dually as both oncogenes and tumor- of cancer.53 Moreover, specific miRNA-expression signa-suppressor genes depending on the cancer type and cellu- tures have been associated with specific translocations inlar context. An example is miR‑125b, which was reported hematopoietic malignancies.54 For example, the fusionto function as either an oncogene or tumor-suppressor gene AML1–ETO produced by the t(8;21) translocationgene in different cancer types or cell lines (Figure 2). In promotes heterochromatic silencing of pre‑miR‑223 inovarian, thyroid, and oral squamous-cell carcinomas, patients with leukemia.55miR‑125b is downregulated and has been shown to Along with the CAGRs harboring miRNA genesinhibit cell proliferation and cell-cycle progression.47,48 and epigenetic changes, several studies have indicatedOn the other hand, miR‑125b inhibited apoptosis in that single nucleotide polymorphisms (SNPs) in bothneuroblastoma cells in a p53-dependent manner 49 and miRNA genes and miRNA-target genes also increasepromoted cell proliferation and invasion in prostate the predisposition to specific types of cancers. Althoughcancer cells.50 A similar duality of function in distinct SNPs are rare in miRNA genes, they can affect miRNAtypes of cancers has also been found for miR‑181a, function in pri-miRNA transcription, pri-miRNA andmiR‑181c, and miR‑220.51 In line with these findings, pre-miRNA processing, and miRNA binding sites. 56it seems that the role of a given miRNA is dependent For example, a SNP found in the binding site of let‑7 inon cancer type and target specificity and, therefore, it is the 3-UTR of the KRAS gene increased the risk of lungimportant to elucidate the role of aberrantly expressed cancer in moderate smokers.57 The presence of the SNPmiRNAs in each type of cancer. rs531564 in pri‑miR‑124‑1 was associated with increasedNATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 8 | AUGUST 2011 | 469 © 2011 Macmillan Publishers Limited. All rights reserved
  4. 4. REVIEWS bladder and esophageal cancer risk.58,59 In breast cancer, cancer (Table 1). Confirming these data, another study SNPs in miRNA genes have been associated with both found that miR‑141 was overexpressed in sera from increased and decreased risk of cancer, such as SNP patients with prostate cancer compared to normal tissue rs11614913 in pre‑miR‑196a‑2,60 and SNP rs895819 in samples. 70 Moreover, 15 serum miRNAs were over- pre‑miR‑27a,61 respectively. SNPs affecting the function expressed in patients with prostate cancer, including of a miRNA can also increase the risk of cancer. The SNP miR‑16, miR‑92a and miR‑92b.70 In a comprehensive rs2910164 located in the 3 strand of miR‑146a promotes study, miRNA-expression profiles were identified in the mispairing in the hairpin of the precursor, altering its sera of patients with lung or colorectal cancer, or diabetes expression and leading to an increased risk of papil- by extracting miRNA from the serum.71 For example, 63 lary thyroid carcinoma.62 Since aberrant expression and new miRNAs that were absent in normal controls were sequence variations in miRNAs are related to cancer risk, detected in the sera of patients with non-small-cell lung it is thought that these noncoding RNAs may function as cancer (NSCLC). Although a unique expression profile useful biomarkers for cancer predisposition. of serum miRNAs was identified for each cancer type, an overlap was found in the profiles of specimens from MiRNAs as biomarkers all diseases analyzed in the study, including diabetes. In Based on the tissue-specific deregulation of miRNA addition, this study also showed that miRNA-expression expression in cancer, multiple studies have explored the profiles differed between the serum and blood cells of potential usefulness of miRNA-expression profiles as bio- lung cancer patients, while similar miRNA-expression markers of cancer diagnosis, prognosis, and response to profiles were seen in the serum and blood cells of healthy treatment. In a study of 143 lung cancer samples from controls. These findings suggest that tumor-specific patients who underwent potentially curative resec- miRNAs in serum are derived not only from circulating tion, patients could be classified into two major groups blood cells but also cancer cells. according to let‑7 expression, with reduced let‑7 expres- Another study in NSCLC demonstrated that a set of sion associated with significantly shorter survival after 11 serum miRNAs were differentially expressed between resection.63 High miR‑21 expression levels were associ- patients with longer or shorter survival, and among this ated with poor survival and therapeutic outcome in 84 set, four (miR‑486, miR‑30d, miR‑1, and miR‑499) were patients with colon adenocarcinoma.64 Expression levels associated with decreased overall survival of patients.72 of miR‑15b, miR‑34c, and miR‑361 may predict a low risk of In a study of epithelial ovarian cancer, eight serum tumor recurrence following curative resection, with an miRNAs, among them miR‑21, miR‑92, miR‑93, miR‑126, overall accuracy of 90% in hepatocellular carcinoma.65 and miR‑29a, were significantly overexpressed in a set In addition, since the loss of specific miRNAs provides a of 19 samples collected before therapy compared with selective advantage for cells destined for metastatic colo- 11 healthy controls.73 This study also provided evidence nization,66 these small RNAs are valuable biomarkers of that miRNAs may be used as biomarkers for early detec- cancer progression and metastasis. The loss of miR‑335 tion by showing that miR‑21, miR‑92, and miR‑93 were and miR‑126 expression in the majority of primary breast overexpressed in three patients with normal levels of pre- tumors in patients who relapse is associated with poor operative cancer antigen 125, a biomarker used for detect- distal-metastasis-free survival.66 In liver cancer, the loss of ing the recurrence of ovarian cancer. In colorectal cancer, miR‑122 expression in tumor cells segregates with speci- a study demonstrated that a set of miRNAs, including fic gene-expression profiles linked to cancer progression miR‑17‑3p and miR‑92, were simultaneously upregulated and gain of metastatic properties.67 Although these find- in plasma and tissue samples.74 By analyzing an indepen- ings suggest the possibility of using small RNAs as bio- dent group of plasma samples, the researchers also markers for noninvasive diagnostic screening and early demonstrated that miR‑92 was differentially expressed in cancer detection, these findings do not yet eliminate the colorectal cancer compared with gastric cancer, inflam- necessity of using invasive cancer screening techniques. matory bowel disease, and tissues from normal controls and may be a potential molecular marker for detect- MiRNA levels in body fluids ing colorectal cancer in plasma samples. Interestingly, Tumor-specific miRNAs were first discovered in the another study also found that the levels of miR‑92a (and serum of patients with diffuse large B-cell lymphoma; of miR‑29a) were significantly higher in plasma samples high levels of miR‑21 correlated with improved relapse- from patients with advanced-stage colorectal cancer than free survival.68 In an elegant experiment in a xenograft in those from healthy controls.75 mouse prostate cancer model, the presence of circulating In a report on gastric cancer, serum levels of upregu- tumor-derived miR‑629 and miR‑660 was confirmed in lated miRNAs such as miR‑21 and miR‑106b were signifi- blood with 100% sensitivity and specificity.69 In addi- cantly higher in patients with gastric cancer than in tion to showing that both serum and plasma samples controls before resection, and were reduced after resec- are adequate for measuring specific miRNA levels, the tion.76 A prospective study reported that tumor-specific investigators reported that by measuring serum levels miRNAs such as miR‑195 were detected and significantly of miR‑141, they were able to distinguish patients with altered in the circulation by using prospectively collected prostate cancer from healthy subjects. Since then, over samples from 127 women, including 83 patients with 100 studies have assessed the potential use of serum breast cancer and 44 healthy age-matched controls. 77 or plasma miRNAs as biomarkers in different types of Furthermore, serum levels of miR‑195 and let‑7a were470 | AUGUST 2011 | VOLUME 8 © 2011 Macmillan Publishers Limited. All rights reserved
  5. 5. REVIEWS Table 1 | A compendium of circulating miRNAs with potential as biomarkers for cancer miRNAs Cancer type Body fluid Healthy Patients Clinical correlations source subjects (n) (n) miRs-21, 155 and 210 Diffuse large B-cell Serum 43 60 High miR-21 expression was associated with relapse-free lymphoma survival68 miR-141 Prostate Serum 25 25 Serum levels of miR-141 distinguished patients from healthy subjects69 miRs-141, 16, 92a, 92b, 103, Prostate Serum 15 6 Serum levels were significantly higher in patients compared 107, 197, 34b, 328, 485-3p, to controls70 486-5p, 574-3p, 636, 640, 766, and 885-5p miRs-486, 30d, 1 and 499 Lung Serum – 243 Serum levels were differentially expressed between patients with longer and shorter survival. The four-miRNA signature was an independent predictor of overall survival72 miRs-21, 92, 93, 126 Ovarian Serum 11 19 miRs-21, 92 and 93 were overexpressed in patients with normal and 29a preoperative cancer antigen 12573 miRs-17-3p and 92 Colorectal Plasma 50 90 Plasma levels decrease after surgery; differentiated colorectal from gastric cancer and normal individuals74 miRs-92a and 29a Colorectal Plasma 59 157 Plasma levels significantly higher in patients with advanced-stage cancer than healthy controls75 miRs-17-5p, 21, 106a Gastric Plasma 69 30 Plasma miRNA levels reflected the tumor miRNAs in most cases; and 106b miRNAs were significantly reduced in post-operative samples76 miR-195 and let7-a Breast Serum 44 83 Serum levels were decreased after tumor resection and correlated with nodal and estrogen-receptor status77 miRs-21, 210, 155, and 196a Pancreas Plasma 36 49 Plasma levels discriminate patients from healthy controls78 miR-210 Pancreas Plasma 25 22 Plasma levels were significantly elevated in two independent patient cohorts79 miR-500 Liver Serum 40 40 Increased levels found in patients with hepatocellular carcinoma; miR-500 serum levels returned to normal after surgical treatment80 miR-206 Rhabdomyo- Serum 17 8 Serum levels of the muscle-specific miRNA miR-206 were sarcoma significantly higher in patients with rhabdomyosarcoma tumors than in patients with other types of tumors or in the control group81 miR-184 Tongue Serum 20 20 Serum levels were significantly reduced after surgical removal of the primary tumors82 miR-92a Acute leukemia Plasma 20 20 Decreased levels in plasma samples of acute leukemia patients84 miRs-125a and 200a Oral squamous-cell Saliva 50 50 Lower levels in the saliva of patients than control subjects86 miR-31 Oral squamous-cell Plasma 21 43 Increased levels in patients compared with controls; level in most Saliva 8 9 patients declined after surgery87 miRs-126, 152 and 182 Bladder Urine 9 47 Increased levels in patients compared with controls88 miR-141 Colorectal Plasma – 102 High levels were associated with poor prognosis123decreased after tumor resection and specific circulating rhabdomyoma also overexpress miR‑206. In patients withmiRNAs were correlated with nodal and estrogen recep- squamous-cell carcinoma of the tongue, plasma levels oftor status. The combined expression analyses of miR‑21, miR‑184 were significantly higher than those in healthymiR‑210, miR‑155, and miR‑196a in plasma can dis- individuals and miR‑184 levels were significantly reducedcriminate pancreatic adenocarcinoma patients from con- after surgical removal of the primary tumors.82trols.78 The plasma levels of the hypoxia-related miRNA Recently, a high-throughput study generated miRNAmiR‑210 were also altered in patients with pancreatic signatures from plasma samples collected 12–28 monthscancer compared to healthy controls from two indepen- before and at the time of lung cancer detection.83 In thisdent cohorts.79 High levels of miR‑500 were found in the study, 21 miRNAs were identified as risk, diag nosis,serum of patients with hepatocellular carcinoma, and and prognosis predictors and are potentially usefulafter tumor resection these levels returned to normal in in the monitoring of high-risk disease-free smokers.three out of 40 patients.80 Serum levels of the muscle- Furthermore, this study is one of the first to demonstratespecific miRNA miR‑206 were significantly higher in that specific pre-disease signatures of miRNA expressionpatients with rhabdomyosarcoma than in patients with in plasma samples can predict the development of lungother types of tumors or those in the control group.81 cancer before diagnosis by conventional techniques andNonetheless, more studies are necessary to identify new in a noninvasive manner.miRNAs as biomarkers in rhabdomyosarcoma because MiRNAs are likely to be useful as noninvasive bio-other rare myogenic tumors such as leoimyosarcoma and markers not only in solid tumors, but also in hematologicNATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 8 | AUGUST 2011 | 471 © 2011 Macmillan Publishers Limited. All rights reserved
  6. 6. REVIEWS malignancies. For instance, in a study in acute leukemias, multivesicular bodies (MVBs) that later fuse with the there was a decrease of miR‑92a in the plasma samples of plasma membrane, releasing the exosomes to the exte- all patients compared with controls.84 A specific profile of rior.95 Exosomes containing miRNAs were found not plasma miRNAs was also found in CLL compared with only in blood,96 but also in other types of body fluids multiple myeloma, hairy-cell leukemia and healthy- such as saliva.97 Interestingly, one group of researchers control samples.85 The results of this study indicated has demonstrated the existence of tumor-derived exo- that circulating miRNAs correlated with the prognosis somes98 and a miRNA signature for circulating ovarian marker ZAP-70 status and might be used to detect and cancer exosomes.99 This miRNA signature was signifi- stratify individuals with CLL. cantly correlated with primary tumor-miRNA expres- Although the majority of studies assessed circulat- sion in women with cancer compared to women with ing miRNAs in serum and plasma, recent studies have benign disease and was not identified in normal controls. confirmed the potential use of tumor-specific miRNAs A similarity between miRNA signatures in circulating as diagnostic markers for cancer in other body fluids. exosomal miRNA and originating tumor cells was also For instance, a study analyzed a panel of four miRNAs in found in lung adenocarcinoma, 100 with a significant the saliva of patients with oral squamous-cell carcinoma difference in exosomal miRNA levels between cancer compared with matched healthy controls; miR‑125a and patients and controls. miR‑200a were present in significantly lower levels in the Importantly, exosomes represent a newly discovered saliva of the cancer patients.86 In another study, miRNA mechanism by which donor cells can communicate and expression was analyzed in the saliva collected in the influence the gene expression of recipient cells. These week before surgery and 6 weeks after surgery from nine findings were first demonstrated by the same study that patients with oral squamous-cell carcinoma and com- discovered miRNAs in exosomes, in which mouse mast pared with eight normal individuals.87 Cancer patients cell exosomes were added to human mast cells, leading to had significantly higher salivary levels of miR‑31 than a subsequent detection of mouse proteins in the human the controls, and eight of nine patients had a decrease in cells.92 Indeed, another study confirmed these findings salivary miR‑31 levels after tumor resection. These results and demonstrated that exosomes released by glioblastoma were also found for miR‑31 plasma levels. Increased levels cells containing mRNA, miRNAs, and angiogenic pro- of miR‑126, miR‑152, and miR‑182 were found in urine teins, such as EGFRvIII, are taken up by normal recipi- samples from patients with bladder cancer, and the ratios ent cells, such as brain microvascular endothelial cells.101 of miR‑126 to miR‑152 and miR‑182 to miR‑152 indicated In this study, it was shown that messages delivered by the presence of bladder cancer with a specificity of 82% tumor-derived exosomes are translated by recipient cells and a sensitivity of 72%.88 to promote tumor progression by stimulating prolifera- MiRNAs have also been detected in other body fluids, tion of a human glioma cell line and tubule formation by such as tears, breast milk, bronchial lavage, colostrum, endothelial cells. In addition, the results indicated that and seminal, amniotic, pleural, peritoneal, and cerebro- cancer patients have elevated levels of tumor-derived exo- spinal fluids.89 Specific compositions and concentrations somes in plasma compared with controls. A recent study, were found for each body fluid analyzed. These find- however, found that a known family of tumor-suppressor ings might be useful if a correlation between specific miRNAs, let‑7, is abundant in exosomes produced by a miRNA levels in body fluids and various disease states metastatic gastric cancer cell line.102 The researchers is proven. suggested that the selective secretion of exosomal let‑7 family members may be related to maintenance of an Body-fluid miRNA stability and activity intercellular tumorigenic and metastatic state. The diagnostic and prognostic potential of miRNAs as Nevertheless, little is known about the mechanisms by cancer biomarkers relies mainly on their high stability which miRNAs are generated in plasma and the biologic and resistance to storage handling. It has been consis- impact of these molecules in distant sites of the body.103 tently shown that serum miRNAs remain stable after In a recent study, the investigators demonstrated that exo- being subjected to severe conditions that would normally somal miRNAs promote gene silencing similar to cellular degrade most RNAs, such as boiling, very low or high miRNAs and that exosomes with miRNAs are released pH levels, extended storage, and 10 freeze–thaw cycles.71 through a ceramide-dependent secretory machinery.104 In addition, recent studies have demonstrated that In this study, it was also demonstrated that the secre- miRNAs are preserved in archived 10-year-old human tion of miRNAs is affected by ceramide levels regulated serum samples,90 and in unrefrigerated dried serum blots by neutral sphingomyelinase 2 (nSMase2) and that the —which may be a more convenient and safer way to save, inhibition of nSMase2 blocked the secretion of miRNAs transport, and store serum and other body fluids, such as and exosomes. In addition, by incubating the metastatic saliva and urine, for miRNA assays.91 This stability can be prostate cancer cell line PC-3M-luc with a conditioned partially explained by the discovery of lipoprotein com- medium from miR‑146a-overexpressing HEK293 cells, plexes, including small membrane vesicles of endocytic the investigators showed an approximately 20% decrease origin called exosomes or microvesicles (30–100 nm), in proliferation. Moreover, the addition of the condi- containing miRNAs, 92 mRNAs, 93,94 and proteins. 94 tioned medium of miR‑146a-transduced COS-7 cells Exosomes can be formed through inward budding significantly knocked down the miR‑146a target gene of endosomal membranes, giving rise to intracellular ROCK1 in PC-3M-luc cells. However, further studies are472 | AUGUST 2011 | VOLUME 8 © 2011 Macmillan Publishers Limited. All rights reserved
  7. 7. REVIEWSnecessary to unveil how miRNAs are sorted into exo- Cytoplasmsomes and whether there is a pathway in which specificmiRNAs are chosen to be incorporated into exosomes. Growing evidence indicates that exosomal miRNApackaging occurs non-randomly based on differentialexpression of exosomal miRNA compared to that of donor Pri-miRNAcells.92 Indeed, studies have demonstrated that nearly 30% Nucleusof the released miRNAs in vitro and in vivo do not reflectthe expression profile found in donor cells, suggesting that MVBspecific miRNAs are selected to be intracellularly retained NPM1or released by exosomes.105 Taken together, these studiesindicate that the secretion of miRNAs by tumor cells is Pre-miRNAassociated with their ability to influence the surround-ing microenvironment for their own benefit. After being dsRNA Ago2transcribed in the nucleus and exported to the cytoplasmin donor cells, pre-miRNA molecules can bind to specificproteins responsible for their stability and associationwith MVBs and exosomes. After fusion with the plasma AA Exosomes AAmembrane, MVBs are able to release exosomes into the AAcirculating compartments and bloodstream. These exo-somes can donate their miRNAs to the recipient cells bythe process of endocytosis. Exosomal miRNAs are pro-cessed by the same machinery used in miRNA biogenesisand thus promote widespread consequences within the HDLcell and lead to an alteration in the physiologic state of thecell. Recently, another mechanism potentially involvingthe nSMase2 pathway was discovered showing that high-density lipoprotein (HDL) transports circulating miRNAsand can alter gene expression by transferring miRNAs torecipient cells (Figure 3).106 This is an exciting finding, butfurther study is necessary to determine the mechanism by Pre-miRNAwhich selective miRNAs are taken up from recipient cellsand how they are released. dsRNA AA Although the presence of miRNAs in exosomes or AA AAHDL could explain their stability in serum, other pos-sibilities include protection by chemical modificationsor association with protein complexes. In this regard, Figure 3 | Biogenesis and mechanism of action of circulating miRNAs. After beingrecent findings showed that the RNA-binding protein transcribed in the nucleus, pre-miRNA molecules can be processed further by Dicernucleophosmin 1 (NPM1) may have a role in the expor- in the cytoplasm. In addition, based on recent findings,92,104–108 there are at least two ways that pre-miRNAs can be packaged and transported using exosomes and MVBstation, packaging, and protection of extracellular miRNAs or other (not fully explored) pathways together with RNA-binding proteins. After fusion(Figure 3).107 Furthermore, recent studies demonstrated with the plasma membrane, MVBs release exosomes into the circulatingthat potentially 90% of the plasma and serum miRNAs compartments and bloodstream. Likewise, pre-miRNA inside the donor cell can beare not encapsulated by vesicles, but cofractionated with stably exported in conjunction with RNA-binding proteins, such as NPM1,107 andprotein complexes. The results indicated that the associ- Ago2,108 or by HDL.106 Circulating miRNAs enter the bloodstream and are taken up byation of Argonaute2 (Ago2; the effector of target mRNA the recipient cells by endocytosis or, hypothetically, by binding to receptors presentsilencing by miRNAs) with plasma and serum miRNAs at the recipient cellular membrane capable of recognizing RNA-binding proteins.influences their stability. Whether the Ago2–miRNA More studies are necessary to elucidate how miRNAs are loaded into exosomes and how they can be internalized by recipient cells. Exosomal miRNAs are processed bycomplex in plasma is capable of regulating the expression the same machinery used in miRNA biogenesis and thus have widespreadof recipient cells is not clear; however, these findings may consequences within the cell by inhibiting the expression of target protein-codingbe useful in the near future for establishing circulating genes. For processing machinery see Figure 1. Abbreviations: MVBs, multivesicularmiRNA as biomarkers.108 bodies; NPM1, nucleophosmin 1; Ago2, Argonaute2; HDL, high-density lipoprotein. It was demonstrated that the cell-free miRNAs wereprobably derived from normal and/or tumor-lysed cellsin body fluids.71,109 Therefore, in order to use miRNAs as mediated by the affected organ or system. There is evi-biomarkers in cancer, it is important to determine the dence that circulating miRNAs in body fluids and extra-source of the tumor-specific miRNAs in body fluids and cellular fluid compartments have hormone-like effects,establish a signature capable of differentiating diseased leading to widespread consequences within the cells atfrom healthy states. Also, it is necessary to clarify whether a distance from the ‘secreting’ cell (Box 1). Nonetheless,the differential expression between tumor and normal additional studies are necessary to elucidate the mecha-tissues is related solely to the tumor or is a response nism by which miRNAs reach the bloodstream andNATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 8 | AUGUST 2011 | 473 © 2011 Macmillan Publishers Limited. All rights reserved
  8. 8. REVIEWS Box 1 | MiRNAs—the ‘oldest’ hormones and differences in blood collection (for example, heparin contains an inhibitor of Taq polymerase).116 Frequently After the seminal discovery of ribozymes, RNAs that perform catalytic functions used reference genes, such as U6 small nuclear RNA in the absence of any DNA or protein molecule, the concept of the RNA world as (RNU6B) and 5S ribosomal RNA, were found to have the primordial world of ‘living’ organisms containing only RNA as genetic material a less-stable expression than others 117 or degraded in is now widely accepted.126 This concept can be extended by considering that the first ‘signaling’ molecules between genomes were also RNAs, and such signals serum samples.71 In addition, the considerable differences were short sequences and very stable, exactly the same as circulating microRNAs in choices of reference genes to use represents a major (miRNAs). As hormones, miRNAs should be released by a donor cell as exosomes obstacle in comparing expression levels between normal or as ‘free’ molecules secreted by active mechanisms and spread signals that tissue and tumors (Table 2). For example, in one study affect cells located in other parts of the organism that uptake the miRNAs either as to identify stable controls for normalization, two of 21 exosomes or as ‘free’ RNAs (Figure 3). Consequently, ‘normal’ levels of circulating miRNAs studied (miR‑142‑3p and miR‑16) were identi- miRNAs vary widely among humans according to age, gender, physiologic events fied as potential ‘normalizers’ given consistent expression (such as menarche or pregnancy) and are influenced by various ‘extrinsic’ factors such as environmental temperature and stress. A practical consequence for this across all patient and control samples.73 The addition of view, is the fact that for any study comparing the expression of miRNAs in any type synthetic versions of miRNAs from other organisms such of body fluid from normal individuals and cancer patients it is important to design as C. elegans in serum and/or plasma samples has proven the study in a ‘paired’ way at least for age, gender and race and use at least twice useful for normalizing the data obtained by qRT-PCR and as many controls as patients. In this way, the ample expression variations in the also may represent an interesting approach to circum- normal population can be assessed and the comparisons with cancer groups will venting normalization issues.69 However, more studies be more meaningful and reproducible in independent cohorts. are necessary for the identification of an accurate nor- malization protocol and empirical validation of stable endogenous control miRNAs for each type of body fluid. Table 2 | Reference genes used to normalize miRNA expression in body fluids Moreover, specific methods have to be standardized for Reference gene Cancer type Body fluid source specimen collection, processing, and purification of total RNA and data analysis, similar to the standard operating miR-16 Diffuse large B-cell lymphoma Serum68 procedures used routinely in laboratories. Synthetic versions of the specific Prostate Serum69 C. elegans microRNAs cel-miR-39, cel-miR-54, and cel-miR-238 Predictors of therapy response Clinical studies have demonstrated the potential for miRs-142-3p and miR-16 Ovarian Plasma73 use of miRNAs as predictors of sensitivity to radio- U6 Colorectal Plasma74 therapy and anticancer agents.118 For instance, the loss miR-16 Gastric Plasma76 of heterozygosity of miR‑128b, an EGFR regulator, was U6, 18S rRNA Breast Serum77 correlated with response to the EGFR inhibitor gefitinib miR-16 and cel-miR-54 Pancreas Plasma78,79 in relapsed patients with NSCLC.119 In colorectal cancer, it was demonstrated that let‑7g and miR‑181b may be miR-16 Liver Serum80 indicators of chemotherapy response to 5-fluorouracil miR-16 Rhabdomyosarcoma Serum81 treatment.120 Likewise, upregulated and downregulated miR-16 Tongue Serum82 miRNAs were detected in sensitive or resistant cell lines miR-638 Acute leukemia Plasma84 and predicted patient response to anticancer agents. One U6 and miR-16 Oral squamous cell Plasma,86 saliva87 study identified a miRNA chemosensitivity profile from a set of 59 human cancer cell lines derived from diverse U6 Bladder Urine88 tissues (NCI-60 cell lines). Downregulation of miR‑34, miR‑17, and let‑7a was related to sensitivity to drugs com- monly used in cancer treatment, such as 5-fluorouracil, the physiologic impact of circulating miRNA in global adriamycin, and cyclophosphamide, respectively.121 These cellular processes. findings suggest that circulating miRNAs may be useful in predicting patterns of resistance and sensitivity to drugs Detection of miRNAs in body fluids used in cancer treatment. Several techniques are currently available for establish- Nonetheless, one study to date has demonstrated a cor- ing miRNA signatures in body fluids, such as miRNA relation between circulating miRNA-expression levels microarrays,70 quantitative real-time PCR (qRT-PCR),68 and response to a given anticancer treatment. In this and deep sequencing (next-generation sequenc- study, serum miR‑21 levels were higher in patients with ing).71 Among these approaches, the most frequently castration-resistant prostate cancer whose disease was used is qRT-PCR and its variations, such as stem-loop resistant to docetaxel-based chemotherapy when com- RT-PCR110,111 and poly(A)-tailed RT-PCR,112 which have pared to those with chemosensitive disease.122 Additional improved the specificity and sensitivity of miRNA detec- and more detailed investigations are needed to gauge the tion.113,114 Nonetheless, most published studies present utility of circulating miRNAs in predicting resistance or conflicting data and have limitations in their cross- sensitivity to specific treatments. Moreover, the study of comparison of miRNA-expression profiles due to differ- circulating miRNAs would provide new insights regard- ing methodologies, various reference genes being used ing the identification of cancer patients responsive to a to normalize the miRNA levels measured in body fluids, specific protocol of anticancer agents before treatment,474 | AUGUST 2011 | VOLUME 8 © 2011 Macmillan Publishers Limited. All rights reserved
  9. 9. REVIEWScircumventing unnecessary treatments and collateral side RB1, TP53, and PTEN), and chromosomal translocations,effects. Furthermore, by identifying specific miRNA sig- together with miRNAs, may also increase the specificitynatures related to cancer progression in body fluids, it may and sensitivity of cancer detection. Third, more studies arebe possible to better determine the efficacy of treatments necessary to establish a standardized and robust methodand select patients for clinical trials. with universal parameters for tumor-specific miRNA detection in body fluids. Finally, such approaches canConclusions be used not only for cancer but for any type of humanMiRNA detection in body fluids is a ‘booming’ field in condition and disease, including highly lethal diseasesthe world of biomarkers and, because more than 1,500 such as septic shock.125,126 From the emerging studies, it istranscribed miRNAs have been identified in the human clear that it is only a matter of time until miRNA markersgenome, miRNA detection could be a potential gold with widespread use and marketability will be identifiedmine for identifying biomarkers, as well as for predict- and confirmed in large cohorts of patients not only withing response to cancer (and to other disease) therapy. cancer, but with many other disorders.However, important issues need to be addressed in orderto establish circulating miRNAs as biomarkers for cancer. Review criteriaFirst, larger prospective clinical trials are needed to validatethese results, since the majority of the published studies The PubMed and Scopus databases were searchedhave small sample sizes and lack long-term outcome for articles using the terms “microRNAs”, “circulating microRNAs”, and “cancer” in combination with “plasma”,data. Second, as miRNAs upregulated or downregulated “serum”, “saliva”, “urine and body fluids”, “stability”,in body fluids are shared by several types of cancer, espe- “treatment”, “predisposition”, “therapy response”,cially ones with common origins, further studies are nec- “chemotherapy”, “detection”, “diagnosis”, “prognosis”,essary to establish a well-characterized panel of miRNAs and “therapy”. Articles published in English betweenspecific to each type of tumor, early or advanced cancer 1993 and 31 January 2011 were considered. The Reviewstage, response to treatment, patient outcome, and recur- also includes one article published in 1980 owing to itsrence.123,124 A combination of known biomarkers, such importance to the field. Searches were also conductedas cancer-related antigens (for example prostate-specific for key authors in this field and the reference lists of identified publications were examined for further material.antigen), mutated genes (for example BRCA1, BRCA2,1. Lee, R. C., Feinbaum, R. L. & Ambros, V. 12. Lee, Y. et al. The nuclear RNase III Drosha 23. He, L. & Hannon, G. J. MicroRNAs: small RNAs The C. elegans heterochronic gene lin-4 encodes initiates microRNA processing. Nature 425, with a big role in gene regulation. Nat. Rev. small RNAs with antisense complementarity to 415–419 (2003). Genet. 5, 522–531 (2004). lin-14. Cell 75, 843–854 (1993). 13. Yi, R., Qin, Y., Macara, I. G. & Cullen, B. R. 24. Bohnsack, M. T., Czaplinski, K. & Gorlich, D.2. Wightman, B., Ha, I. & Ruvkun, G. 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