Extracellular microRNA: A new source of biomarkers.
Etheridge A, Lee I, Hood L, Galas D, Wang K.
Mutat Res. 2011 Mar 23
Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103, United States.

MicroRNAs (miRNAs) are a recently discovered class of small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes, including differentiation, neoplastic transformation, and cell replication and regeneration. Because of these regulatory roles, it is not surprising that aberrant miRNA expression has been implicated in several diseases. Recent studies have reported significant levels of miRNAs in serum and other body fluids, raising the possibility that circulating miRNAs could serve as useful clinical biomarkers. Here, we provide a brief overview of miRNA biogenesis and function, the identification and potential roles of circulating extracellular miRNAs, and the prospective uses of miRNAs as clinical biomarkers. Finally, we address several issues associated with the accurate measurement of miRNAs from biological samples.

Circulating microRNAs: Association with disease and potential use as biomarkers.
Reid G, Kirschner MB, van Zandwijk N.
Critical Reviews in Oncology/Hematology (2010)
Asbestos Diseases Research Institute (ADRI), Bernie Banton Centre, University of Sydney, Gate 3, Hospital Road, Concord, Sydney, NSW 2139, Australia.

The control of gene expression by microRNAs influences many cellular processes and has been implicated in the control of many (patho)physiological states. Recently, microRNAs have been detected in serum and plasma, and circulating microRNA profiles have now been associated with a range of different tumour types, diseases such as stroke and heart disease, as well as altered physiological states such as pregnancy. Here we review the disease-specific profiles of circulating microRNAs, and the methodologies used for their detection and quantification. We also discuss possible functions of circulating microRNAs and their potential as non-invasive biomarkers.

Analysis of circulating microRNA - preanalytical and analytical challenges.
McDonald JS, Milosevic D, Reddi HV, Grebe SK, Algeciras-Schimnich A.
Clin Chem. 2011 57(6): 833-840
Department of Laboratory Medicine and Pathology, MayoClinic, Rochester, MN 55905, USA.

BACKGROUND: There is great interest in circulating microRNAs (miRNAs) as disease biomarkers. Translating promising miRNAs into validated clinical tests requires the characterization of many preanalytical and analytical parameters.
METHODS: miRNAs were extracted from serum and plasma samples of healthy volunteers, and miRNAs known to be present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were amplified by reverse-transcription quantitative PCR. Stability and the effects of hemolysis were determined. Assay variation and its components, including the effect of adding control miRNA, were assessed by nested ANOVA.
RESULTS: miRNA concentrations were higher in plasma than in serum. Processing of plasma to remove subcellular/cellular components reduced miRNA concentrations to those of serum. The miRNAs analyzed were stable refrigerated or frozen for up to 72 h and were stable at room temperature for 24 h. Hemolysis increased the apparent concentration of 3 of the miRNAs. The total variability of replicate miRNA concentrations was <2.0-fold, with most of the variability attributable to the extraction process and interassay imprecision. Normalizing results to those of spiked exogenous control miRNAs did not improve this variability.
CONCLUSIONS: Detailed validation of the preanalytical steps affecting miRNA detection and quantification is critical when considering the use of individual miRNAs as clinical biomarkers. Unless these causes of imprecision are considered and mitigated, only miRNAs that are extremely up- or downregulated will be suitable as clinical biomarkers.

Circulating microRNAs: promising breast cancer biomarkers
Helen M Heneghan, Nicola Miller* and Michael J Kerin
Breast Cancer Research 2011, 13:402

We read with interest the recent article by Roth and colleagues [1] reporting the fi ndings of altered tumor-specifi c microRNAs (miRNAs) in sera of breast cancer patients. Th is report further substantiates emerging data suggesting that blood-based miRNAs have immense potential as novel non-invasive cancer biomarkers. However, we have several concerns regarding the authors’ study.Roth and colleagues claim this article to be the fi rst evidence that circulating miRNAs have potential as breast cancer biomarkers, yet refer to previous reports of similar fi ndings [2,3]. Th is aside, other claims in the study are unsubstantiated. Firstly, the fi nding that total RNA levels were signifi cantly higher in M0 patients compared to controls and M1 breast cancer patients most likely refl ects the quality of RNA extraction techniques and is not clinically relevant. We have previously demonstrated that total RNA levels diff er signifi cantly depending on the RNA isolation method and starting blood medium [2]. Th e authors have not adequately discussed their fi nding that patients with metastatic disease had signifi cantly lower total RNA levels compared to M0 patients; if their claim that total RNA concentration indicated tumour progression held truth, then one would expect a sequential increase in total RNA concentration from controls, to M0 and M1 patients..........................

Intracellular and extracellular microRNAs in breast cancer.
Corcoran C, Friel AM, Duffy MJ, Crown J, O'Driscoll L.
Clin Chem. 2011 57(1): 18-32
School of Pharmacy and Pharmaceutical Sciences and Molecular Therapeutics for Cancer Ireland (MTCI), Trinity College Dublin, Dublin, Ireland.

BACKGROUND: Successful treatment of breast cancer is enhanced by early detection and, if possible, subsequent patient-tailored therapy. Toward this goal, it is essential to identify and understand the most relevant panels of biomarkers, some of which may also have relevance as therapeutic targets.
METHODS: We critically reviewed published literature on microRNAs (miRNAs) as relevant to breast cancer.
SUMMARY: Since the initial recognition of the association of miRNAs with breast cancer in 2005, studies involving cell lines, in vivo models, and clinical specimens have implicated several functions for miRNAs, including suppressing oncogenesis and tumors, promoting or inhibiting metastasis, and increasing sensitivity or resistance to chemotherapy and targeted agents in breast cancer. For example, miR-21 is overexpressed in both male and female breast tumors compared with normal breast tissue and has been associated with advanced stage, lymph node positivity, and reduced survival time. miR-21 knock-down in cell-line models has been associated with increased sensitivity to topotecan and taxol in vitro and the limitation of lung metastasis in vivo. Furthermore, the discovery of extracellular miRNAs (including miR-21), existing either freely or in exosomes in the systemic circulation, has led to the possibility that such molecules may serve as biomarkers for ongoing patient monitoring. Although additional investigations are necessary to fully exploit the use of miRNAs in breast cancer, there is increasing evidence that miRNAs have potential not only to facilitate the determination of diagnosis and prognosis and the prediction of response to treatment, but also to act as therapeutic targets and replacement therapies.

Serum microRNAs as non-invasive biomarkers for cancer.
Brase JC, Wuttig D, Kuner R, Sültmann H.
Mol Cancer. 2010 9: 306.
Working Group Cancer Genome Research, German Cancer Research Center, Heidelberg, Germany

Human serum and other body fluids are rich resources for the identification of novel biomarkers, which can be measured in routine clinical diagnosis. microRNAs are small non-coding RNA molecules, which have an important function in regulating RNA stability and gene expression. The deregulation of microRNAs has been linked to cancer development and tumor progression. Recently, it has been reported that serum and other body fluids contain sufficiently stable microRNA signatures. Thus, the profiles of circulating microRNAs have been explored in a variety of studies aiming at the identification of novel non-invasive biomarkers. In this review, we discuss recent findings indicating that circulating microRNAs are useful as non-invasive biomarkers for different tumor types. Additionally, we summarize the knowledge about the mechanism of microRNA release and the putative functional roles of circulating microRNAs. Although several challenges remain to be addressed, circulating microRNAs have the potential to be useful for the diagnosis and prognosis of cancer diseases.

Cell-free nucleic acids as biomarkers  in cancer patients
Heidi Schwarzenbach, Dave S. B. Hoon and Klaus Pantel
Nat Rev Cancer. 2011 11(6): 426-437
Institute of Tumour Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.

DNA, mRNA and microRNA are released and circulate in the blood of cancer patients. Changes in the levels of circulating nucleic acids have been associated with tumour burden and malignant progression. In the past decade a wealth of information indicating the potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies has emerged. In this Review, we discuss these findings with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers.

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients.
Mostert B, Sieuwerts AM, Martens JW, Sleijfer S.
Expert Rev Mol Diagn. 2011 11(3): 259-275
Daniel den Hoed Cancer Center, Laboratory of Clinical Tumor Immunology, Rotterdam, The Netherlands.

Recently, miRNA-expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation probably has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Circulating tumor cells (CTCs) have rapidly developed as important prognostic and therapy-monitoring biomarkers in metastatic breast, colorectal and prostate cancer when enumerated, and their isolation enables subsequent analysis using various molecular applications, including miRNA-expression analysis. In addition to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs. In this article, we will discuss miRNA-expression profiling in primary tumors, depict the potential applications of measuring miRNA in the circulation and review the literature on cell-free circulating miRNAs, as well as offering some methodological and technical considerations on the measurement of circulating miRNAs.

Identification of muscle-specific microRNAs in serum of muscular dystrophy animal models: promising novel blood-based markers for muscular dystrophy.
Mizuno H, Nakamura A, Aoki Y, Ito N, Kishi S, Yamamoto K, Sekiguchi M, Takeda S, Hashido K.
PLoS One. 2011 6(3): e18388
Administrative Section of Radiation Protection, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMD(J)), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMD(J). Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy.

Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer.
Hu Z, Chen X, Zhao Y, Tian T, Jin G, Shu Y, Chen Y, Xu L, Zen K, Zhang C, Shen H.
J Clin Oncol. 2010 28(10): 1721-1726
Department of Epidemiology and Biostatistics, Cancer Center, Nanjing Medical University, 140 Hanzhong Rd, Nanjing 210029, China.

PURPOSE: Recent findings that human serum contains stably expressed microRNA (miRNA) have revealed a great potential of serum miRNA signature as disease fingerprints to predict survival. We used genome-wide serum miRNA expression analysis to investigate the role of serum miRNA in predicting prognosis of non-small-cell lung cancer (NSCLC).
PATIENTS AND METHODS: To control disease heterogeneity, we used patients with stages I to IIIa lung adenocarcinoma and squamous cell carcinoma, who were treated with both operation and adjuvant chemotherapies. In the discovery stage, Solexa sequencing followed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays was used to test the difference in levels of serum miRNAs between 30 patients with longer survival (alive and mean survival time, 49.54 months) and 30 patients with shorter survival matched by age, sex, and stage (dead and mean survival time, 9.54 months). The detected serum miRNAs then were validated in 243 patients (randomly classified into two subgroups: n = 120 for the training set, and n = 123 for the testing set).
RESULTS: Eleven serum miRNAs were found to be altered more than five-fold by Solexa sequencing between longer-survival and shorter-survival groups, and levels of four miRNAs (ie, miR-486, miR-30d, miR-1 and miR-499) were significantly associated with overall survival. The four-miRNA signature also was consistently an independent predictor of overall survival for both training and testing samples.
CONCLUSION: The four-miRNA signature from the serum may serve as a noninvasive predictor for the overall survival of NSCLC.

The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform.
Resnick KE, Alder H, Hagan JP, Richardson DL, Croce CM, Cohn DE.
Gynecol Oncol. 2009 112(1): 55-59

OBJECTIVE: To determine the utility of serum miRNAs as biomarkers for epithelial ovarian cancer.
METHODS: Twenty-eight patients with histologically confirmed epithelial ovarian cancer were identified from a tissue and serum bank. Serum was collected prior to definitive therapy. Fifteen unmatched, healthy controls were used for comparison. Serum was obtained from all patients. RNA was extracted using a derivation of the single step Trizol method. The RNA from 9 cancer specimens was compared to 4 normal specimens with real-time PCR using the TaqMan  Array Human MicroRNA panel. Twenty-one miRNAs were differentially expressed
between normal and patient serum. Real-time PCR for the 21 individual miRNAs was performed on the remaining 19 cancer specimens and 11 normal specimens.
RESULTS: Eight miRNAs of the original twenty-one were identified that were significantly differentially expressed between cancer and normal specimens using the comparative C(t) method. MiRNAs-21, 92, 93, 126 and 29a were significantly over-expressed in the serum from cancer patients compared to controls (p<.01). MiRNAs-155, 127 and 99b were significantly under-expressed (p<.01). Additionally,
miRs-21, 92 and 93 were over-expressed in 3 patients with normal pre-operative CA-125.
CONCLUSION: We demonstrate that the extraction of RNA and subsequent identification of miRNAs from the serum of individuals diagnosed with ovarian cancer is feasible. Real-time PCR-based microarray is a novel and practical means to performing high-throughput investigation of serum RNA samples. miRNAs-21, 92 and 93 are known oncogenes with therapeutic and biomarker potential.

Circulating microRNAs as stable blood-based markers for cancer detection.
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M.
Proc Natl Acad Sci U S A. 2008 105(30): 10513-10518

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
Supporting information - Circulating microRNAs as stable blood-based markers for cancer detection.

Serum microRNAs are promising novel biomarkers.
Gilad S, Meiri E, Yogev Y, Benjamin S, Lebanony D, Yerushalmi N, Benjamin H, Kushnir M, Cholakh H, Melamed N, Bentwich Z, Hod M, Goren Y, Chajut A.
PLoS One. 2008 5;3(9): e3148.

BACKGROUND: Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers.
METHODS AND RESULTS: After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage.
CONCLUSIONS AND SIGNIFICANCE: Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

Detection of cancer with serum miRNAs on an oligonucleotide microarray.
Lodes MJ, Caraballo M, Suciu D, Munro S, Kumar A, Anderson B.
PLoS One. 2009 4(7): e6229.

Micro RNAs (miRNAs) are a class of small, non-coding RNA species that play critical roles throughout cellular development and regulation. miRNA expression patterns taken from various tissue types often point to the cellular lineage of an individual tissue type, thereby being a more invariant hallmark of tissue type. Recent work has shown that these miRNA expression patterns can be used to classify tumor cells, and that this classification can be more accurate than the classification achieved by using messenger RNA gene expression patterns. One aspect of miRNA biogenesis that makes them particularly attractive as a biomarker is the fact that they are maintained in a protected state in serum and plasma, thus allowing the detection of miRNA expression patterns directly from serum. This study is focused on the evaluation of miRNA expression patterns in human serum for five types of human cancer, prostate, colon, ovarian, breast and lung, using a pan-human microRNA, high density microarray. This microarray platform enables the simultaneous analysis of all human microRNAs by either fluorescent or electrochemical signals, and can be easily redesigned to include newly identified miRNAs. We show that sufficient miRNAs are present in one milliliter of serum to detect miRNA expression patterns, without the need for amplification techniques. In addition, we are able to use these expression patterns to correctly discriminate between normal and cancer patient samples.

Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.
Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY.
Cell Res. 2008 18(10): 997-1006.

Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

A truth serum for cancer - microRNAs have major potential as cancer biomarkers.
Chin LJ, Slack FJ.
Cell Res. 2008 18(10): 983-984.

Identification of biological markers of cancer is a major area of research. Biomarkers could identify the presence of a tumor before it could otherwise be easily detected, and the ability to detect cancers at early stages is a key factor in increasing survivability. For example, the American Cancer Society finds that a reason breast cancer survival rates are so high is that there are good methods for early detection of tumors. However, this is not the case for most cancers. For lung cancer, the five-year survival is 15%, but for the 16% of lung cancer cases diagnosed at early stages, the five-year survival rate is 49% 1. While this is just one example, the ability to identify a cancer while it is still localized is clearly beneficial. Currently, most methods for discovering and testing tumor biomarkers are difficult and labor-intensive procedures, and at most, only several markers can be tested for at one time. However, due to the simplicity of getting a blood sample, easily testable biomarkers found in blood serum would be especially useful.
Just recently, scientists have begun identifying microRNAs (miRNAs) as cancer biomarkers 2, 3, 4. MiRNA genes code for a relatively new class of regulatory RNAs that are ~22 nucleotides long. miRBase (Release 12.0), the central database for miRNAs, lists over 8 000 miRNAs from plants, animals and viruses. Many miRNAs are well conserved across species, suggesting an important role for them. MiRNA biogenesis and regulation of gene expression have been extensively covered in numerous reviews 5, 6, 7. Briefly, most mature miRNAs are the products of RNA polymerase II-transcribed transcripts that have been processed by two RNase III enzymes, Drosha and Dicer. The mature miRNA is incorporated into an RNA-induced silencing complex that binds to a target messenger RNA (mRNA). In animals, miRNAs bind with imperfect complementarity to the 3′ untranslated region of their targets to inhibit gene expression through several possible mechanisms, including degradation of the mRNA, inhibition of the initiation or elongation steps of translation, and localization to cytoplasmic P-bodies. Because animal miRNAs bind with imperfect complementarity, miRNAs are though to be capable of targeting numerous mRNAs; thus, misexpression of one miRNA can disrupt the expression of hundreds of proteins......................

Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma.
Lawrie CH, Gal S, Dunlop HM, Pushkaran B, Liggins AP, Pulford K, Banham AH, Pezzella F, Boultwood J, Wainscoat JS, Hatton CS, and Harris AL.
Br J Haematol. 2008 141(5): 672-675.

Circulating nucleic acids have been shown to have potential as non-invasive diagnostic markers in cancer. We therefore investigated whether microRNAs also have diagnostic utility by comparing levels of tumour-associated MIRN155 (miR-155), MIRN210 (miR-210) and MIRN21 (miR-21) in serum from diffuse large B-cell lymphoma (DLBCL) patients (n = 60) with healthy controls (n = 43). Levels were higher in patient than control sera (P = 0.009, 0.02 and 0.04 respectively). Moreover, high MIRN21 expression was associated with relapse-free survival (P = 0.05). This is the first description of circulating microRNAs and suggests that microRNAs have potential as non-invasive diagnostic markers for DLBCL and possibly other cancers.

Profiling of microRNA in Blood Serum/Plasma
Guidelines for the miRCURY LNATM Universal RT microRNA PCR System

by Exiqon 2011

Introduction:  MicroRNA profiling in serum and plasma samples holds great promise as a non-invasive way to discover important new biomarkers for a wide range of diseases and biological  processes . However, getting microRNA profiles from such samples can be challenging .The miRCURY LNA™ Universal RT microRNA PCR system offers the sensitivity and specificity needed for performing accurate microRNA expression profiling from serum and plasma samples (Figure 1) . This document contains a set of guidelines for setting up microRNA profiling experiments from blood serum and plasma and provides important information and tips to ensure successful microRNA quantification using the miRCURY LNA™ Universal RT microRNA PCR system .

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