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.
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
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,
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.
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.
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
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.
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.
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.
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 
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 . 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
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,
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
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.
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,
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.
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.
of cell-free and circulating tumor cell-associated miRNAs in cancer
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.
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
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,
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.
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.
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).
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
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.
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.
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
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
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.
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
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.
information - Circulating microRNAs as stable blood-based markers for
microRNAs are promising
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.
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.
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
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.
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.
(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
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.
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.
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.
truth serum for
cancer - microRNAs have major potential as cancer biomarkers.
Chin LJ, Slack FJ.
Cell Res. 2008 18(10): 983-984.
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
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......................
of elevated levels of
tumour-associated microRNAs in serum of patients with diffuse large
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.
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.
microRNA in Blood Serum/Plasma
Guidelines for the miRCURY LNATM Universal RT microRNA PCR System
by Exiqon 2011
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 .