review article micrornas as potential biomarkers...

Review ArticleMicroRNAs as Potential Biomarkers in CancerOpportunities and Challenges

Huiyin Lan1 Haiqi Lu2 Xian Wang2 and Hongchuan Jin1

1Laboratory of Cancer Biology Institute of Clinical Science Sir Run Run Shaw Hospital School of MedicineZhejiang University Hangzhou Zhejiang 310020 China2Department of Medical Oncology Sir Run Run Shaw Hospital School of Medicine Zhejiang UniversityHangzhou Zhejiang 310020 China

Correspondence should be addressed to Hongchuan Jin jinhczjueducn

Received 6 June 2014 Accepted 5 September 2014

Academic Editor Francesco Nicassio

Copyright copy 2015 Huiyin Lan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

MicroRNAs (miRNAs) are a group of small noncoding RNAs (ncRNAs) that posttranscriptionally regulate gene expression bytargeting their correspondingmessenger RNAs (mRNAs) DysregulatedmiRNAs have been considered as a new type of ldquooncomiRsrdquoor ldquotumor suppressorsrdquo playing essential roles in cancer initiation and progression Using genome-wide detection methodsubiquitously aberrant expression profiles ofmiRNAshave been identified in a broad array of human cancers showing great potentialas novel diagnostic and prognostic biomarkers of cancer with high specificity and sensitivity The detectable miRNAs in tissueblood and other body fluids with high stability provide an abundant source for miRNA-based biomarkers in human cancersDespite the fact that an increasing number of potential miRNA biomarkers have been reported the transition of miRNAs-basedbiomarkers from bench to bedside still necessitates addressing several challenges In this review we will summarize our currentunderstanding of miRNAs as potential biomarkers in human cancers

1 Introduction

According to NIH (National Institutes of Health) a bio-marker is described with a characteristic that it is objectivelymeasured and evaluated as an indicator of normal biologicalprocesses pathogenic processes or pharmacologic responsesto a therapeutic intervention [1] Useful biomarkers canprovide great insights into tumorigenesis and facilitate thedevelopment of improved therapies Cancer is not only oneof the biggest global killers but also one of the fastestgrowing causes of death [2] Hunting for cancer biomarkerscapable of providing diagnostic prognostic or therapeuticinformation has become a necessary but challenging workin cancer research Currently some available biomarkerssuch as PSA (prostate-specific antigen) for prostate cancerCEA (carcinoembryonic antigen) for colorectal cancer AFP(a-fetoprotein) for liver cancer CA 125 for ovarian cancerand CA 19ndash9 for pancreatic cancer invariably display aninferior sensitivity and specificity despite their generallyclinical applications Due to high rates of ldquooverdiagnosisrdquo and

ldquopseudodiseaserdquo by PSA test the USPSTF (US PreventiveServices Task Force) issued a grade D recommendation torecommend against routine PSA-based screening for prostatecancer in July 2012 [3] Therefore it is an urgent request formore reliable biomarkers to serve as precise indicators ofdevelopments and changes of cancer at the cellular levels andthe patientrsquos response of cancer therapy

The discovery of miRNAs has captured increased atten-tion of researchers recently Following the Lin-4 inC elegansknown as the first member of miRNAs discovered in 1993[4] more and more miRNAs have been found and inves-tigated herein a number of miRNAs have been identifiedto essentially revolve in human carcinogenesis by acting asldquooncoMirsrdquo or ldquotumor suppressive miRNAsrdquo [5] Strikinglybased on the pervasive cancer-associated aberrant expressionprofiles of dysregulated miRNAs together with the charac-teristics of miRNAs such as temporal and spatial specificitysensitivity and stability in both paraffin sections and bodyfluids miRNAs have shown great potential as a new class ofbiomarkers in cancer Furthermore with the extensive use

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 125094 17 pageshttpdxdoiorg1011552015125094

2 BioMed Research International

of next generation of technologies such as miRNA microar-rays and high-throughput deep sequencing techniques [6]translating biomarker into practice with increased diagnosticand therapeutic sensitivity and specificity would be less of aproblem [7] Among these contexts the potential of miRNAsbeing involved in carcinogenesis as cancer biomarkers hasbeen extensively investigated and developed

In this review we will overview recent advances in thedysregulation of tumor-associated miRNAs in both biopsiesand circulation and highlight some critical challenges for thetransition of miRNAs as cancer biomarkers from a researchsetting into clinical application

2 Overview of miRNAs DefinitionBiogenesis and Functions

miRNAs belong to the heterogeneous class of ncRNAsof 22ndash24 nt RNAs that play important regulatory roles bytargeting mRNAs for cleavage or translational repression [8]By definition miRNAs are small RNA molecules incapableof encoding proteins but possessing important structuralcatalytic and regulatory functions According to the latestmiRBase release (v21 June 2014 httpwwwmirbaseorg)there are 28645 entries representing hairpin precursor miR-NAs from 223 species expressing 35828 mature miRNAproducts MiRNA genes are located either in independentnoncoding DNA loci or in the introns of protein-codinggenes [9] Furthermore up to 50 miRNAs are clusteredon chromosomes with a common promoter [10] and cotran-scribed to form different miRNAs families which sharecommon mRNAs targets and biological processes because oftheir identical seed regions [11]

It is well established that miRNA biogenesis is a com-plex process which generally includes three main steps asfollows (i) In the nucleus miRNA genes are transcriptedby RNA polymerase II to form primary miRNA transcripts(pri-miRNAs) [12] (ii) Subsequently the Drosha RNase IIIendonuclease trims the pri-miRNA to liberate a pre-miRNAhairpin which is actively transported to the cytoplasm byRan-GTP and exportin-5 [8 13] (iii) Its final matura-tion is processed in the cytoplasm where Dicer RNase IIIendonuclease cleaves the pre-miRNA into a single-strandedmature miRNA [13] and then the mature miRNA binds toproteins of the argonaute (Ago) family and assembles theRNA induced silencing complex (RISC) together to exert itsfurther physiological functions

After being incorporated into the RISC the maturemiRNA induces posttranscriptional gene silencing by teth-ering RISC to be partially complementary to the targetmRNA predominantly found within the 31015840-untranslatedregion (UTR) [14] Inspiringly a recent study showed that asingle miRNAmay repress more than 100mRNAs on averageand over 60 of human protein-coding genes are conservedtargets of miRNAs [15] Given this vast majority of mRNAtargets regulated by miRNAs aberrant miRNA expressionprofoundly influences a wide variety of cell regulation path-ways important to cell proliferation [16] apoptosis [17] andstress responses [18] In cancer disease phenotype miRNAs

serve exclusively as a novel class of oncogenes or tumorsuppressor genes named ldquooncomiRsrdquo or ldquotumor suppressivemiRsrdquo [19] Following the first identified tumor suppressivemiRNA genemiR-15amiR-16-1 revolving in the developmentof B-cell lymphocytic leukemia [20] a growing number ofoncomiRs have been validated as well as tumor suppressivemiRs (more details are in the review [21]) Remarkablyit has been continually confirmed by numbers of studiesthat ldquooncomiRsrdquo or ldquotumor suppressive miRsrdquo revolve in amultitude of cancer-related signaling pathways For examplemiR-17 activates PI3KAKT pathway and regulates tumorgrowth [22] the axis of p53-miRNA-34 network regulatesWnt pathway [23] and promotes EMT program [24] andlet-7 leads to growth inhibition by negatively regulatingRAS pathway [25] Taken together these observations givea miRNAs-mediated complex network model that connectsoncogenic and tumor suppressive signaling pathways inhuman cancers indicating that miRNAs may play a moreindispensable role in the pathogenesis of human cancers thanpreviously thought

3 miRNAs as Potential Biomarkers in Cancer

31 Aberrant miRNA Expression Profiles in Cancer

311 Linkage of Aberrant miRNA Expression Profiles withCancer Development Aberrant expression of miRNAs incancer is characterized by abnormal expression levels ofmature or precursor miRNA transcripts in comparison withthose in the corresponding normal tissues A steadily growingnumber of reports have proven that human cancers fre-quently show an aberrant expression profile of miRNAs aswell as pre-miRNAs With various kinds of high-throughputsequencing platforms applied to analyze the genome-wideexpression of miRNA genes these years aberrant expressionprofiles ofmiRNAs either downregulated or upregulated havebeen discovered in a broad variety of human malignant can-cers including lymphoma [26] breast cancer [27] colorectalcancer [28] prostate cancer [29] and glioma [30]

Although the causes of aberrant miRNAs expression incancer have not yet been fully elucidated several definedmechanisms can be possible including chromosomal abnor-malities genomic mutations epigenetic changes and alter-ations in miRNA biogenesis Notably some miRNA genesare frequently located at fragile sites and genomic regionsrelated to cancer [31] In other words alterations of miRNAsexpression can directly reflect the chromosomal or genomicchanges of cancer-associated genes Together with importantroles of cancer-associatedmiRNAs identified in various typesof cancer cell lines [32] and clinical tumor specimens [33ndash35] the aberrant expressions of miRNAs appear to showsignificant values of clinical applications

312 Diagnostic Prognostic and Predictive Values of miRNASignatures for Cancer Given those circumstances abovealterations ofmiRNAs expression in different types of neopla-sia constitute specific patterns ofmiRNA signature for certaintypes of cancer Logically these specific miRNA signatures

BioMed Research International 3

would be attractively conducive to early diagnosis signifyingthe prognosis and predicting the response of cancer therapy

(i) Diagnosis and Classification It was observed that withcancers derived from different epithelial and hematopoieticlineage having distinct miRNA profiles miRNA signatureswere able to discriminate them according to their devel-opmental origins For example in a research trying toidentify cancers with unknown primary tissue of origin byusing microarray platform to evaluate the expressions of 47miRNAs in 101 FFPE (formalin fixed and paraffin embedded)samples from primary or metastatic cancers overall theaccuracy reached 100 for primary cancers and 78 formetastatic cancers When the signature was applied to anindependent published dataset of 170 samples correct predic-tion was further verified in 86 of the metastasis cases [36]

For patients with malignant cancers precise determina-tion of tumor subtype significantly influences the decision-making of treatments For example In renal cell carcinoma(RCC) a research team developed a classification systemapplying method of decision trees that can distinguish dif-ferent RCC subtypes in 94 different subtype cases usinguniquemiRNA signatures analysis [37]This system obtaineda sensitivity of 97 for differentiating normal cases fromRCC patients And among the four types of RCC (clearcell RCC (ccRCC) papillary RCC (pRCC) oncocytoma andchromophobe RCC (chRCC)) 100 for ccRCC subtype fromthe other fours 97 for pRCC subtype from oncocytoma andchRCC and 100 accuracy for distinguishing oncocytomafrom chRCC subtype [37] Moreover another study by Giladet al validated a miRNA-based assay capable of differentiat-ing between fourmain types of lung cancer in an independentset of 451 samples They got a result for more than 90 ofthe samples with overall accuracy of 94 with a similarperformance observed in pathologic and cytologic samples[38] Collectively these findings above suggested that miR-NAs signatures were able to serve as an accurate classificationtool for different cancers in pathologic and cytologic samples

Moreover the need of biomarkers for cancer early diag-nosis is extremely important because of the fact that survivaland prognosis of patients extensively depend on the stage oftumor at the time of detection with an early diagnosis gener-ally associated with a better prognosis Promisingly miRNAsignatures have been identified to possess a robust potentialas early diagnosis biomarkers For example overexpressionof miR-205 and miR-21 in ductal adenocarcinoma has beenreported to precede phenotypic changes in the ducts [39]suggesting that aberrant miRNA production was an earlyevent in the development of cancer and thus supporting thepossibility to use them for early detection of cancers

(ii) Prognostic Values In addition to cancer diagnosis includ-ing distinguishing tissue of origin subtypes and early detec-tion of cancermiRNA signatures can be also valuable for can-cer prognosis prediction which has been vilified by a numberof studies recently The first evidence was proposed by Calinet al in chronic lymphocytic leukemia (CLL) where a uniquemiRNA signature composed of 13 genes can differentiateCLL patients with abnormal expression of prognostic factors

(ZAP-70 and IgV(H)) from normal cases Besides the samemiRNAsignaturewas associatedwith the presence or absenceof disease progression [40]Moreover in lung cancer a signa-ture of five miRNAs has been used in the prediction of treat-ment outcome for non-small-cell lung carcinoma (NSCLC)among which high expression of miR-221 and let-7a isassociated with good prognosis as opposed to elevated miR-137miR-372 andmiR-182with poor prognosis [41] Likewisehigh expression of themiR-183 family (miR-183miR-182 andmiR-96) was associated with overall poor survival in patientswith lung cancer [42] In addition a recent study interrogatedplasmamiR-10b andmiR-373 levels which are knownmedia-tors ofmetastasis in breast cancer and they uncovered associ-ation of thesemiRNAs for detecting the lymph nodemetasta-sis thereby showing potential as prognostic biomarkers [43]It is worth noting that one miRNA alone can also possessaccurate predictive power which was clarified by a studyin patients with breast cancer they identified the fact thatoverexpression ofmiR-210 is associatedwith an increased riskof recurrence and a reduced chance of relapse-free survivaland miR-210 alone allowed prediction of prognosis to thesame level as a 76-gene mRNA signature test (GENE76) [44]In prostate cancer miRNAs have also been connected toprognosis of PC such as miR-141 and miR-375 which turnedout to correlate with high Gleason score or lymph-nodepositive status in a second independent validation study [45]Besides signature of miR-410 and miR-645 was negativelyassociated with overall survival in advanced serous ovariancancer [46] Remarkably a pattern of seven-miRNA signaturerecently identified in gastric cancer can predict overall sur-vival and relapse-free survival [47] and in patients with stageII colon cancer a six-miRNA-based classifier established wasan independent prognostic factor for disease recurrence [48]

(iii) Predictive Values for Response to Therapy Apart fromtheir use as prognostic biomarkers miRNA signatures couldalso predict the responses to various cancer managementsThe correlation betweenmiRNA signatures and the responsesof specific therapies is of great clinical value because ofthe involvement of miRNAs in chemoresistance identifiedin many studies [49 50] For instance Meng et al firstlyfound that inhibition of miR-21 and miR-200b increasedsensitivity to gemcitabine in cholangiocarcinoma cell lines[51] Subsequently it was proven that high miR-21 expressioninvolved in gemcitabine chemoresistance was able to predictsignificantly shorter overall survival (OS) in pancreatic can-cer treated with gemcitabine [52] Likewise by conferringtaxol resistance through the suppression of Bak1 expressionhigh expression of miR-125b in breast cancer predicted thepoor response to taxol-based treatments [53] In colorectalcancer the presence of the KRAS mutation was associatedwith upregulation of miR-127-3p miR-92a and miR-486-3pand downregulation of miR-378 which constituted a miRNAsignature capable of predicting colorectal cancers resistantto EGFR antagonists [54] Additionally it was demonstratedthat let-7g and miR-181b may be indicators of chemotherapyresponse to S-1 based chemotherapy in CRC [55] In gliomacancer miR-181d upregulation may correspond to a betterresponse to temozolomide while upregulation ofmiR-21may


predict poor response caused by temozolomide resistance[30] Remarkably in relapsed patients with NSCLC theloss of heterozygosity of miR-128b an EGFR regulator wascorrelated with response to the EGFR inhibitor gefitinib [56]In belief given these specific miRNA signatures serving aspredictors of response to various cancer management it maybe possible to improve patientsrsquo prognosis by selecting theright treatment course as soon after diagnosis as possibleand permit rapid adaption of treatment to the acquisition ofchemotherapeutic and radioresistance What is more is thatby predicting response to molecular-targeted agents the useof predictive biomarkers could be promisingly conducive toaccelerate the development of new anticancer therapies inclinical trials [57]

In summary based on the aberrant miRNA expressionprofiles identified in human cancers miRNAs give us a totallynew horizon in understanding the developmentmanagementof cancer Despite of some certain miRNAs commonlyexhibiting altered expression across various kinds of tumorsparticular types of cancers often express their exclusivepatterns of miRNAs referring to their tissues of origin [33]Such specific miRNA signatures exhibited in types of tumorsmentioned above are also termed as ldquomiRNomerdquo whichcharacterizes the malignant state and defines some of theirclinicopathological features such as grade stage aggressive-ness vascular invasion and proliferation index [31] Giventhe great value of miRNA signatures for cancer diagnosisprognosis and prediction of therapeutic responses a com-prehensive list of miRNAs identified as various biomarkersin different cancer types are summarized in Table 1

32 Potential Source of miRNA-Based Biomarkers

321 Stability of miRNAs In addition to the high specificityand sensitivity of miRNA biomarkers in cancer diagnosis aspreviously described miRNAs are characterized by robuststability as well in stored RNA samples including fixed tissueblood and other body fluids which further supports theirpotential as biomarkers Recent studies have systematicallyinvestigated the stability of miRNAs even after being sub-jected to severe conditions that would normally degrademRNA or other types of larger RNAs For example in twosimilar studies miRNA expression profiles in FFPE fromcolorectal cancer (CRC) samples dating back up to 10 yearwere assessed and compared with fresh frozen CRC samplesa high similarity of miRNAs profiles was observed More-over differing formalin fixation times did not significantlyinfluence miRNAs profiles in FFPE specimens in light ofquantitative real-time polymerase chain reaction (qRT-PCR)[58 59] For another instance of extracellular miRNAs stabil-ity reported by Chen at al they treated some miRNAs withor without RNase A digestion then they surprisingly foundthat half ofmiRNAs remain intact after 3 hours of exposure toRNase A but all high molecular weight RNAs set as controlswere rapidly degraded More importantly after being treatedwith several harsh conditions including boiling lowhigh pH(pH = 113) extended storage or freeze-thaw cycles miRNAsin sera still maintained potent intrinsic stability as expected[60] Taken together these data consistently revealed that

miRNAs particularly serum miRNAs possess considerableresistance to the enzymatic cleavage by RNase A and varioussevere conditions so as tomaintain their intrinsic stability forlong

Generally there are mainly three explanations for miR-NAsrsquo stability Firstly as a class of small molecules witha short length of only 20ndash24 nt miRNAs tend to be lesssubjected to degradation during FFPE sample processingSecondly these small RNAs are taken into encapsulation bycell-derived lipid vesicles such as exosomes [61] micropar-ticles [62] or apoptotic bodies [63] However the precisemechanisms of miRNAsrsquo selective packaging remain elusiveyetThirdly the nuclease-resistant extracellularmiRNAswerepredominantly associated with RNA binding proteins such asargonaute proteins (Ago1 or Ago2) nucleophosmin (NPM1)or ribosomal proteins [64] which exert a robust protectiveeffect on miRNAs from abundant nucleases in extracellularspace Overwhelmingly Turchinovich et al argued that mostof the extracellular miRNAs are not exclusively associatedwith cell-exported exosomes instead a majority of themare floating outside exosomes to bind to Ago2 protein [64]Nevertheless no final conclusion has yet been reached on themain mechanism of how to maintain miRNAsrsquo stability Butwhat we can confirm is that inherent stability of miRNAs inboth FFPE and circulation perfectly presents great advantagesfor them as ideal biomarkers of cancer

322 miRNAs in FFPET Tissue biopsy specimen in theform of FFPET (FFPE tissue) is commonly used for tissuestorage which preserves good tissue integrity so as to pro-vide excellent resources for discovery of disease-detectingbiomarkers not excepting the miRNAs-based biomarkers ofcancer certainly Better than mRNAs which are too unstableto be suitable for qRT-PCR miRNAs are stable enough tobe well preserved even over prolonged FFPE block storageand quantified from FFPET by qRT-PCR In a word miRNAsfrom substantial FFPET can provide tremendous source forcancer-related biomarkers

Numerous studies have discovered and evaluated a broadvariety of potential miRNAs biomarker candidates in FFPETwhich provide valuable information for cancer diagnosisand prognosis A recent study used bioinformatics to screenmiRNAs classifiers in 69 FFPE pancreatic specimens includ-ing benign premalignant and malignant Different miRNApanels mapped to these lesions above separate anyone fromthe two otherswith sensitivity and specificity up to 85ndash100[65] Furthermore in another study by Casanova-Salas et al[66] an independent cohort of 273 FFPET samples was usedfor validation of new potential biomarkers by qRT-PCRTheyassessed the relationship of two miRNAs (miR-182 and miR-187) most differentially expressed in prostate cancer with theclinicopathological characteristics and outcome of patientsand it was observed that miR-187 expression was decreasedin advanced prostate cancer cases which was consistent withmicroarray data Moreover the higher expression of miR-182independently conferred a worse prognosis for BPFS and PFSusing Cox proportional hazard multivariable analysisThere-fore such results showed the promising potential of miRNAsfor serving as biomarkers in cancer diagnosis or prognosis


Table1Ty

peso

fspecific

miRNAs

biom

arkersin

different

cancers

Cancer

Type

ofbiom

arker

miRNAs

biom

arkers

Reference

Breastcancer

Diagn

ostic

miR-10b-21-30a-92a-125b-141-145-200-801-155-191-203-210

[112113]

Progno

stic

miR-10b-373miR-210

[4344

]Predictiv

emiR-125b

[53]

Lung

cancer

Diagn

ostic

miR-21-155m

iR-16miR-17-19b-25-29c-30c-106a-126-451-66

0-28-3p

[114ndash116]

Progno

stic

miR-221let-7a-137m

iR-372-182

[4142]

miR-1-15b-16-21-126-142-3p-148a-197

[116]

Predictiv

emiR-128b

[56]

Liverc

ancer

Diagn

ostic

miR-222-223-181a-181b-181c-200c-203-21-224-10b-222126-96

[117118]

Progno

stic

miR-21-22-26-29-31-122-124-135a-139-145-146

a-155-200c-221-222-222-223

[119]

Predictiv

emiR-21-200b

[52]

Colon

cancer

Diagn

ostic

miR-15b-18a-29a-335miR-17-3p-20a-21-92-601-760-29a

[120121]

Progno

stic

miR-141

[122]

Predictiv

emiR-127-3p-92a-486-3p-378let-7

gmiR-181b

[5455]

Pancreaticcancer

Diagn

ostic

miR-205-21m

iR-642b-885-5p-22

[39123]

miR-145-150-223-636-26b-34a-122-126lowast-145-150-223-505-636-8855p

[124]

Progno

stic

miR-130bmiR-21-105-196a-2-203-210-222-452-105-127-187-5

18a-2-3

0a-3p

[125126]

Predictiv

emiR-21

[52]

Prostatecancer

Diagn

ostic

miR-30c-622-1285miR-10b-373let-7

c-7e

[43127]

miR-141-375miR-26a-195

[128]

Progno

stic

miR-141-375miR-20a-21-141-145-221

[45129]

Predictiv

emiR-21

[130]

Ovaria

ncancer

Diagn

ostic

miR-200

familylet-7

familym

iR-21-29a-92-93m

iR-126-127-132-155-214-182-205-144-222-302

[131ndash133]

Progno

stic

miR-410-64

5miR-200

familym

iR-141-429

[46134]

Predictiv

emiR-23a-27a-30clet-7g-199a-3p-181a-181b-213

[132]


Table 2 miRNAs as potential biomarkers in FFPE samples

Cancer miRNAs as biomarkers Expression Reference

Glioblastoma

miR-323 -329 -155 -210 Up [135]miR-326 -130a Down [135]miR-21 -221 Up [136]miR-128 -181b Down [136]

Head and neck cancer

miR-21 -31 -107 -138 -504 -10b Up [137]miR-16 -20a -106b -142-3p Up [138]miR-155 -423 -451 let-7i Up [138]miR-10a -125b -375 Down [138]

Breast cancermiR-21 -29b-2 -125b Up [139]miR-16 -155 -191 -196a Up [139]miR-34b -145 -205 let-7a Down [140 141]

Lung cancer miR-21 -205 Up [142]miR-16 Up [115]

Liver cancermiR-519d Up [143]miR-146a Down [144]miR-185 Down [145]

Gastric cancermiR-21 -221 -222 Up [146]miR-10b -21 -106a -223 -338 Up [47]miR-30a-5p -31 -126 let-7a Down [47]

Pancreatic cancer

miR-192-5p -202-3p -337-5p -130-3p Up [65]miR-187 -30a-3p Up [147]miR-21 -155 -203 -210 -222 let-7a Up [148]miR-200c Down [149]

Cervical cancer miR-21 -27a -34a -146a Up [150]miR-155 -196a -203 -221 Up [150]

Ovarian cancer miR-223 Up [151]miR-9 -200a -200b -429 Down [151]

Prostate cancer

miR-31 -143 -221 Up [152]miR-126 -146a -150 Down [152]miR-182 Up [66]miR-187 Down [66]miR-203 Down [153]

Colorectal cancer miR-31 -135b Up [154]miR-21 -23a -193a-3p -338-5p Down [155]

BladdermiR-10a-5p -31-5p -130a -3p Up [156]miR-145 -30a-3p -125b -133a Down [157]miR-133b -195 -199a Down [157]

In addition other miRNAs identified as potentialbiomarkers in FFPET for various types of malignancies aresummarized in Table 2

323 miRNAs in Blood (Total Blood Serum Plasma andExosome) Apart from the diagnostic miRNA signaturesidentified in specimens from tumor tissues studies have alsoshown the diagnostic and prognostic usefulness of miRNAsin circulation In fact except for miRNAs in the biopsiesand tumor samples a substantial number of cancer-relatedmiRNAs can be also detected in blood including total bloodserum and plasma as well as other body fluids such as salivaurine breast milk seminal fluid and tears [67]

In fact the intriguing phenomenon of cancer-associatednucleic acids in circulation has been identified for decadesGenerally from their intracellular origin miRNAs can besecreted extracellularly in cell-derived extracellular vesiclessuch as exosomes or bound to other lipoproteins as men-tioned above although the concrete mechanisms for theorigin of such miRNAs released by tumor cells have not yetbeen clarified and little is known about the mechanisms ofhow to specifically direct miRNAs into multivesicular bodiesor exosomes

However what we know at present is that miRNAs inblood released by tumor cells may not simply act as a set ofbystander product of tumor cells It has beenwidely identified


that tumor-released (TR) exosomes could act as intercellularmediators transporting diverse types of proteins mRNA andmiRNAs that can promote angiogenesis cell proliferationand cell survival resulting in establishing an oncogenicniche systemically [68] Recently a paradigm of autocrineand paracrine miRNA signaling pathway contributing tomicroenvironment of cancer through TR exosomes has beenproposed For example Skog et al firstly found that themessengers encoding for EGFRvIII protein and miR-21 aretransported in glioblastoma-derived exosomes and thesemolecules could be taken up by normal host cells and trans-formed into functional signals stimulating proliferation ofcancer cells [69] Furthermore Fabbri recently demonstratedthat miR-21 and -29a secreted by tumor cells via exosomescould bind to toll-like receptor (TLR) 78 and activate thesereceptors on immune cells leading to TLR-mediated NF-120581B activation and secretion of prometastatic inflammatorycytokines that may ultimately lead to tumor growth andmetastasis [70] These observations above revealed that themobile miRNAs entrapped in exosomes in blood couldfunction as hormones entering the circulation and travellingto distant organs to exert their hormone-like effects leadingto widespread consequences within the recipient cells at adistance from the donor cells

Additionally Pigati and colleagues verified the fact thatthe cancer-related miRNAs are specific to the cancer cellswhich is a further consideration supporting detection of thecirculating miRNAs to serve as molecular tumor markersThey found that the bulk of miR-451 and miR-1246 pro-duced by malignant cells was selectively released into bloodparticularly but the majority of these miRNAs produced bynormal cells were retained intracellularly [71] Their findingsindicated that release of miRNAs into blood and otherbiological fluids is selective presumably with an explanationthat the assortment of them might be changed by malignanttransformation [72]

Therefore with a stable form and extensively hormone-like effects the noninvasively detectable cancer-specificmiRNAs in circulation have been widely investigated anddemonstrated to efficiently discriminate cancer patients fromhealthy people predict the prognosis and monitor thetherapeutic response Comprehensively recent studies fordiscovery of potential miRNAs biomarkers for types of can-cers in blood and other body fluids have been listed in Table 3

In serum following the first paradigm showed thatthe diagnostic and prognostic potential of miR-21 that wasassociated with increased relapse-free survival of diffuse largeB-cell lymphoma (DLBCL) patients by Lawrie et al [73]and lots of new candidate miRNA biomarkers in serum ofcancer patients have been reported For example Chen et aldemonstrated that a profiling of 10 differentially expressedserum miRNAs selected from a sample set including 400NSCLC cases and 220 controls provided a high sensitivityand specificity for NSCLC diagnosis They used risk scoreanalysis to evaluate the diagnostic value of the serummiRNAsprofile system and found that these 10 miRNAs were ableto distinguish NSCLC cases from controls with sensitivityof 93 and specificity of 90 What is more is that these10 serum miRNAs were correlated with the stage of NSCLC

patients [74] Moreover in a recent study Wu et al reported35miRNAs targeting 11 genes involved in theTGF-120573 signalingpathway for their associations with survival among 141 highlyexpressed serum miRNAs collected from 391 patients withadvanced NSCLC They concluded that 17 miRNAs weresignificantly associatedwith 2-year patient survival usingCoxregression model Of these 17 miRNAs miR-16 exhibitedthe most statistical significance whose high expression wasassociated with an obviously better survival [75] Findings ofsimilar crosstalk between TGF-beta signaling and miRNAsmachinery also have been clarified in other cancers such asCRC [76] and glioblastoma [77]

In plasma Mitchell et al firstly proved that miRNAsare readily measurable by direct cloning and sequencingfrom a plasma small RNA library To further determinewhether tumor-derived miRNAs enter the circulation atlevels sufficient to be measurable as biomarkers they estab-lished a mouse prostate cancer xenograft model systemcollected the plasma and isolated miRNAs for analyzing thedifferent expression of miRNAs compared to the controlThey found that levels of miR-629 and miR-660 were able toindependently differentiate xenografted mice from controlswith 100 sensitivity and 100 specificity [78] Such con-clusion thereby provided a firm grounding of the fact thatthat tumor-derived miRNAs measured in plasma can serveas noninvasion biomarkers for cancer detection Examplesare numerous Plasma miR-21 miR-145 and miR-155 usedin combination helped in distinguishing lung cancer fromhealthy smokers with 694 sensitivity and 783 specificity[79] Combination of miR-148b miR-409-3p and miR-801 discriminated powerfully between breast cancer casesand healthy controls [80] Three plasma microRNAs (miR-106b miR-20a and miR-221) had a statistically significantelevation in gastric cancer patients so as to serve as novelbiomarkers for the early detection of GC [81] Interestinglythe combination of miR-16 miR-196a and CA19-9 was moreeffective for pancreatic cancer diagnosis especially in earlytumor screening [82] Ng et al revealed that expression ofmiR-92 in plasma is significantly elevated and differentiatedCRC from gastric cancer or normal subjects of CRC patientswith a high sensitivity of 89 and specificity of 70 thusmiR-92 in plasma could have roles as a potential markerfor CRC screening [68] Moreover high expression of miR-27b miR-148a and miR-326 was associated with decreasedprogression-free survival of CRC patients [68]

Exosomes are a class of small (50ndash90 nm) membranevesicles formed in the plasma membrane by the fusionof endocytic vesicles and extracellularly released into sur-rounding body fluids [83] As is mentioned above miRNAspassively released from normal or tumor-lysed cells maybe preferentially secreted and packaged into exosomes thatprovide a protective effect on the stability of extracellularmiRNAs [61] Excitingly it was discovered that the exoso-mal miRNAs content was similar to that in the originaltumors [84] thus peaking researchersrsquo interests in the use ofexosomal miRNA profiles for diagnostic markers of cancerFor example Hessvik compared miRNAs in PC-3 cellswith the noncancerous prostate cell line by microarray andqRT-PCR analysis It was found that the exosomal miRNA


Table 3 Potential miRNAs biomarkers for types of cancers in blood and other body fluids

Cancer Samples miRNAs biomarkers Expression Reference

GlioblastomaSerum miR-15b -23a -133a -150 -197 -497 Up [158]Plasma miR-128 -342-3p Down [30]CSF miR-21 -17-5p -200 Up [30]

Head and neckPlasma miR-31 -10b -24 -181 -184 Up [87]

Saliva miR-31 Up [87]miR-200a -125a Down [87]

Breast cancer SerummiR-373 -155 Up [159]miR-34a -17 Down [159]miR-222 -103 -23a -29a -23b -24 -25 Up [160]

Plasma miR-148b -376c -409-3p -801 Up [80]

Lung cancer

Serum miR-21-3p -205-5p -205-3p -141 -200c Up [92]miR-21 -24 -205 -30d Up [93]

Plasma miR-21 -155 Up [114]miR-145 Down [114]

Exosome miR-17-3p -21 -106a -146 -155 -191-192 -203 -205 -210 -212 -214 Up [161]Sputum miR-205 -210 -708 Up [90]

Liver cancer Serum

miR-122 Up [162]miR-500 Up [163]miR-21 -122 -223 Up [164]miR-21 -1 -25 -92a -206 -375 let-7f Up [165]miR-16 Down [166]

Gastric cancer

Serum miR-221 -376c -744 Up [167]miR-1 -20a -27a -34 -423-5p Up [168]

PlasmamiR-106b -20a -221 Up [81]miR-21 -223 -218 Up [169]miR-451 -486 Down [170]

Pancreatic cancerSerum miR-21 -100 -155 Up [171]

miR-203 -369-5p -376a -375 Up [172]Plasma miR-21 -210 -155 -196a Up [82 173]Exosome miR-21 -17-5p Up [174]

Colorectal cancer

Serum miR-29a Up [175]

Plasma

miR-17-3p -92 Up [68]miR-92a -29a Up [176]miR-27b -148a -326 Up [177]miR-221 Up [178]miR-15b -17 Up [179]

Ovarian cancer

Serum miR-21 -92 -93 -126 -29a Up [131]miR-155 -127 -99b Down [131]

Plasma miR-205 Up [180]let-7f Down [180]

Exosome miR-21 -141 -200abc -203 -205 -214 Up [84]

Prostate cancer Serum

miR-26a-1 -141 -375 Up [181]miR-16 -195 -26a let7i Up [128]miR-20b -874 -1274a -1207-5p -93 -106a Up [182]miR-223 -26b -30c -24 Down [182]

Exosome miR-107 -574-3p Up [85]

Bladder

BloodmiR-129 -133b Up [183]miR-26b-5p -144-5p -374-5p Up [184]miR-378g -942 -106a-5p -142-3p -374a Up [185]

Urine

miR-18a -25 -187 Up [89]miR-142-3p -140-5p -204 Down [89]miR-214 Up [91]miR-200 -155 -192 -205 Up [186]


Table 3 Continued


Large B-cell lymphoma SerummiR-21 -155 -210 Up [73]miR-15a -16-1 -29c -155 Up [187]miR-34a Down [187]

Leukemia SerummiR-181b-5p -10a-5p -93-5p Up [188]miR-129-5p -155-5p -320d Up [188]miR-29c -223 Down [189]

profile released by PC-3 was highly similar to the profileof corresponding parent cells In a study of ovarian cancerresearchers found that levels of 8 exosomal miRNAs (miR-21 miR-141 miR-200a miR-200c miR-200b miR-203 miR-205 and miR-214) previously demonstrated as diagnosticmarkers were similar between cellular and exosomal miR-NAs which were significantly distinct from profiles observedin benign disease [84] In another study exosomal miRNAswere profiled from the plasma of prostate cancer patientswith or without metastases and a distinct set of 11 miRNAswas present at significantly greater amounts in patientswith metastases compared to those without metastases Andthe association of miR-141 and miR-375 among these 11microRNAs was confirmed in plasma exosomes from a sep-arate patient cohort with recurrent or nonrecurrent diseasethus demonstrating that changes of miRNAs concentrationpresent in exosomes could be of great value for early detectionand tumor staging diagnosis [85] Collectively these resultssuggested thatmiRNAprofiles of circulating tumor exosomescould potentially be used as surrogate diagnostic markers forbiopsy profiles and could also serve as potential diagnosticand prognostic biomarkers of cancer

324 miRNAs in Other Body Fluids Although the major-ity of studies identified circulating miRNAs in serum andplasma miRNAs have also been detected in other bodyfluids such as urine tears breast milk and seminal amni-otic bronchial lavage as well as pleural peritoneal andcerebrospinal fluids [67 86] Recent studies have confirmedthe potential use of tumor-specific miRNAs as diagnosticor prognostic markers for cancer in these body fluidsFor instance miR-125a and miR-200a were identified withsignificantly lower levels in the saliva for oral cancer detection[87] The RNA ratio of miRNA-126 miRNA-152 enabled thedetection of bladder cancer from urine with high specificityof 82 and sensitivity of 72 [88] A subset of six miRNAs(miR-187 miR-18alowast miR-25 miR-142-3p miR-140-5p andmiR-204) was capable of correctly classifying bladder urothe-lial cell carcinoma (UCC) patients with an overall sensitivityof 848 and specificity of 865 [89] In combination ofmiR-205 miR-210 and miR-708 in sputum distinguishedlung squamous cell carcinoma patients from normal subjectswith 73 sensitivity and 96 specificity thus helping inthe early detection of lung squamous cell carcinomas [90]What is more is that urinary level of cell-free miR-214 couldbe an independent prognostic parameter for non-muscle-invasive bladder cancer recurrence [91] Presence of miR-21

and miR-15b in cerebrospinal fluid (CSF) of glioma patientsalso has been identified as a source of biomarkers [30]

However as a matter of fact miRNAs no matter inserum plasma exosomes or other body fluids may not beabsolutely specific to a certain cancer type MiR-21 in serumis exactly a representative example for this From a serial ofinvestigations it is found that high expression of serummiR-21 in lung cancer associated with poor prognosis [92] and thedynamic change of serummiR-21 between postoperative andpreoperative lung carcinoma patients can potentially serveas biomarkers for disease recurrence after surgery operation[93] In other types of cancer also the level of serum miR-21 significantly elevated in patients with CRC and advancedadenoma thus exhibiting its potential diagnostic value forearly detection of CRC [94] as well as the prognostic valuebecause of the postoperative reductions in serummiR-21 levelwhich occurred [95] Observations of analogous diagnosticand prognostic significance also have been identified inesophageal squamous cell carcinomas [96] pancreatic cancer[97] hepatocellular carcinoma [98] and ovarian cancer [99]Therefore a concept that has to be noted again is theldquomiRNomerdquo defined as the full complement of miRNAs in acell which emphasizes that different tissues have distinctivepatterns of miRNome expression with each tissue presentinga specific signature [100] In order tomap to every type of can-cer with higher accuracy and specificity further studies withlarger sample numbers are needed to allow generalized detec-tion of unique patterns of miRNome toward every type ofcancer even before or after therapy and thus may really helpin the identification of useful biomarkers in cancer efficiently

4 Methods for MiRNA Detection

Though miRNAs show striking potential as new biomarkersof human cancers in clinic settings and maintain highstability in both FFPET and body fluids the methods formiRNAsrsquo detection are still challenging yet for the intrinsiccharacterisitic of miRNAs such as small size low level ahigh degree of sequence similarity among various membersand tissue-specific or stage-specific expressions Currentlythere are various strategies for the detection of miRNAsincluding qRT-PCR in situ hybridization (ISH) enzymaticluminescence miRNA assay and northern blotting microar-rays deep sequencing or next-generation sequencing (NGS)and nanopore technology et al [101] Herein qRT-PCR isthe most commonly usedmanner quantifying themiRNomefrom low levels with high sensitivity and specificity which is


very important for the lack of large amounts of RNA fromclinical samples could be obtained More importantly qRT-PCR is more convenient and reliable than methods based onhybridization for its potential as it can be performed in high-throughput assays to allow for large-scale miRNAs detectionat the same time and still with high specificity or sensitivity[102] Therefore qRT-PCR technique has become the goldstandard of nucleic acid quantification currently [103] Never-theless several limitations of qRT-PCR also hinder its use inpractice For example the selection of inner reference genes isessential and critical for data normalization but the determi-nation of suitable reference genes as the normalizer remainsas a highly controversial issue As previous studies displayedthe commonly used reference genes includemiR-16miR-142-3p 18S rRNA miR-638 let-7a miR-1249 miR-295 5SRNAU6 U6B RNU38B RNU43 RNU62 and SNORD43 [104]Unfortunately there is no single gene that is constitutivelyexpressed in all cell types and under all experimental con-ditions [105] Rigorous experimental justification for thesedata reported is deficient and these data are contradicted andinconsistent in their crossing comparison so none of themhas been widely accepted as a standard control Thereforefurther studies are needed to develop a set of stable internalcontrol genes for accurate quantification of miRNAs profilesby qRT-PCR even for each type of human cancers

Microarray is another technique for miRNAs profilingbased on nucleic acid hybridization between target moleculesand their corresponding complementary probes The miR-NAs oligonucleotide probes are immobilized to glass slidesthrough covalent crosslinking between the amino groups andthe SAM (self-assembling monolayer) forming a ready-to-use miRNA microarray When hybridized with the miRNAmicroarray the isolatedmiRNAs labeled with fluorescent dyecan emit fluorescence after being specifically bound to thecorresponding probes Through detecting the fluorescencesignal intensity at different positions on the slides relativequantities of the corresponding miRNAs can be evaluatedconsequently [106] Furthermore microarray-based tech-niques are particularly attractive for their powerful high-throughput function allowing for detecting the presence of awide range of definedmiRNAs simultaneouslywithin numer-ous samples processed in parallel in a single experimentHowever deficiencies of microarray also exist for examplebackground noise and crosshybridization problems as wellas measuring only the relative abundances of previouslydiscovered miRNAs [107]

Deep sequencing relying on next-generation sequenc-ing machines appears to be another promising quantitativetechnology for miRNAs profiling because it provides high-throughput sequencing enabling processing up to millionsof sequence reads in parallel simultaneous with high speed[108] In this method adapters are ligated to the preparedcDNA libraries from RNA sample of interest followed bythe massively parallel sequencing of millions of individualcDNA molecules from the library Bioinformatic analysistranslates the sequence reads into miRNAs abundance levelswith relative quantification and then discovers novelmiRNAsdifferentially expressed and their associated genes by usingdigital approaches may be publicly available or custom-made

software tools [107] Compared with the microarray deepsequencing technologies are not subject to the problemsof background noise and crosshybridization they mea-sure absolute abundance over a wider dynamic range thanmicroarrays and are not limited by array content allowingfor reflecting the actual picture of the miRNA profiles andthe discoveries of novel miRNAs having not been previouslyidentified [109]

Nanopore single-molecule technology a novel methodrecently reported by several studies to electrically detectmiRNAs in tissue or biofluids requires no labeling enzymereactions or amplification [110 111] Wang et al showedthat a nanopore sensor which generates a target-specificsignature signal by using a programmable oligonucleotideprobe were capable of quantifying subpicomolar levelsof cancer-associated miRNAs and distinguishing single-nucleotide differences betweenmiRNA familymembers [111]Coinciding with the work of Wang et al another study byWanunu et al used a 3 nm synthetic nanopore to detect thecomplex of livermiR-122a hybridized with the probemiRNAduplex enriched through binding to the viral protein p19and then the abundance of the duplex was quantified usinga nanopore providing accurate quantification for cellularmiRNAs in tissues finally [110] Taken together these worksby them demonstrated that the nanopore-based miRNAstechniques may with promising potential for quantitativemiRNAs detection to discover more novel biomarkers andthus it would be validated as noninvasive method for earlydiagnosis in cancer patients

5 Perspectives and Challenges

Years of research work have indicated that miRNAs playimportant regulatory roles in translational repression even-tually contributing to the cancer development Indeed geneprofiling studies have identified a number of significant dys-regulatedmiRNAs as ldquooncomiRsrdquo or ldquotumor suppressorsrdquo in avariety of human cancers and overwhelming amounts of datahave strongly validated that the ubiquitously aberrant expres-sions of miRNAs were closely associated with the pathogen-esis and progression of human malignancies These dysreg-ulated miRNAs constitute unique patterns of miRNome indifferent categories of cancers which provide accurate finger-prints to detect and classify various cancers Particularly ascirculatingmiRNAs in blood and other body fluids are readilydetected by various techniques in a relatively noninvasivemanner the clinical application of miRNAs as a new genera-tion of cancer biomarker is considered to be prospecting

Despite the encouraging results we are still confrontedwith many difficulties on the long way of transiting miRNAsas biomarker from bench to bedside First exactly as theproposing of the misconceptions that the 98 in humangenome is noncoding and thus junk DNA miRNAs areprobably only the tip of the iceberg of noncoding genes inour complex genome More miRNA transcripts remain tobe excavated extensively Second more fundamental inves-tigations are needed to further elucidate the exact miRNAsrsquofunctional roles in cancer biology Apart from the pathway bytargeting mRNAs for cleavage or translational repression if


there are any other unrevealedmechanisms led to the transla-tional repression or even any other undiscovered regulationfunctions more than posttranscriptional regulation Thirdit is needed to further clarify the potential biological func-tions of circulating miRNAs particularly the mechanismsof MVBs-contained miRNAs for cell-cell communicationtumor immune evasion and tumormicroenvironment Afterall the underlying mechanism of miRNAs secretion or themechanisms of the selectivity of miRNAs packaging are stillelusive Fourth more consistent and reliable miRNA signa-tures or miRNome in both FFPET and circulation are urgentto be established by adequately large sample size of cohortstudies onmultiple independent populations Fifthmethodsfor the detection and analysis of miRNAs should be furtheroptimized To ensure the miRNAsrsquo detection results are asconsistent as possible a set of technicalmethods bothmiRNAextraction procedures and technology platforms should becompared with each other or optimized for different types ofcancer patients In addition it is worth noting that furtherstudies are needed to develop a set of stable inner referencegenes to accurately quantify miRNAs even for each type ofhuman cancers

Despite such challenges that are a lot the potential ofmiRNAs as a new class of cancer biomarkers is attractiveand cannot be underestimated MiRNAs are being expectedto bring an exciting new dimension to the field of clinicalmanagement for human cancers in the near future

Conflict of Interests

No potential conflicts of interests were disclosed regardingthe publication of this paper

Authorsrsquo Contribution

Huiyin Lan and Haiqi Lu contributed equally to the work

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (nos 81301706 81372178) Department ofHealth in Zhejiang Province (WKJ2012-2-013 2013RCA029)andZhejiangProvincialNatural Science Foundation ofChina(nos Y13H160010 and LR12H16001)

References

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[2] R Lozano M Naghavi K Foreman et al ldquoGlobal and regionalmortality from 235 causes of death for 20 age groups in 1990and 2010 a systematic analysis for the Global Burden of DiseaseStudy 2010rdquoThe Lancet vol 380 no 9859 pp 2095ndash2128 2012

[3] V A Moyer ldquoScreening for prostate cancer US preventive ser-vices task force recommendation statementrdquo Annals of InternalMedicine vol 157 no 2 pp 120ndash134 2012

[4] R C Lee R L Feinbaum and V Ambros ldquoThe C elegansheterochronic gene lin-4 encodes small RNAs with antisensecomplementarity to lin-14rdquoCell vol 75 no 5 pp 843ndash854 1993

[5] Y W Kong D Ferland-McCollough T J Jackson and MBushell ldquomicroRNAs in cancer managementrdquo The LancetOncology vol 13 no 6 pp e249ndashe258 2012

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[7] C Wilson S Schulz and S A Waldman ldquoBiomarker devel-opment commercialization and regulation individualizationof medicine lost in translationrdquo Clinical pharmacology andtherapeutics vol 81 no 2 pp 153ndash155 2007

[8] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[9] J Krol I Loedige and W Filipowicz ldquoThe widespread reg-ulation of microRNA biogenesis function and decayrdquo NatureReviews Genetics vol 11 no 9 pp 597ndash610 2010

[10] V N Kim J Han and M C Siomi ldquoBiogenesis of small RNAsin animalsrdquo Nature Reviews Molecular Cell Biology vol 10 no2 pp 126ndash139 2009

[11] C P Concepcion C Bonetti and A Ventura ldquoTheMicroRNA-17-92 family ofMicroRNA clusters in development and diseaserdquoCancer Journal vol 18 no 3 pp 262ndash267 2012

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[14] M D Jansson and A H Lund ldquoMicroRNA and cancerrdquoMolec-ular Oncology vol 6 no 6 pp 590ndash610 2012

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[81] H Cai Y Yuan Y-F Hao T-K Guo X Wei and Y-M ZhangldquoPlasma microRNAs serve as novel potential biomarkers forearly detection of gastric cancerrdquo Medical Oncology vol 30article 452 no 1 2013

[82] J Liu J Gao Y Du et al ldquoCombination of plasma microRNAswith serum CA19-9 for early detection of pancreatic cancerrdquoInternational Journal of Cancer vol 131 no 3 pp 683ndash691 2012

[83] G van Niel I Porto-Carreiro S Simoes and G Raposo ldquoExo-somes a common pathway for a specialized functionrdquo Journalof Biochemistry vol 140 no 1 pp 13ndash21 2006

[84] D D Taylor and C Gercel-Taylor ldquoMicroRNA signatures oftumor-derived exosomes as diagnostic biomarkers of ovariancancerrdquo Gynecologic Oncology vol 110 no 1 pp 13ndash21 2008

[85] R J Bryant T Pawlowski J W F Catto et al ldquoChanges incirculating microRNA levels associated with prostate cancerrdquoBritish Journal of Cancer vol 106 no 4 pp 768ndash774 2012

[86] Y Ge L Xiao X Chen Y Peng L Sun and F Liu ldquoMicroR-NAs in peritoneal dialysis effluent are promising biomarkersfor peritoneal fibrosis in peritoneal dialysis patientsrdquo MedicalHypotheses vol 78 no 1 pp 155ndash156 2012

[87] N J Park H Zhou D Elashoff et al ldquoSalivary microRNAdiscovery characterization and clinical utility for oral cancerdetectionrdquo Clinical Cancer Research vol 15 no 17 pp 5473ndash5477 2009

[88] M Hanke K Hoefig H Merz et al ldquoA robust methodology tostudy urine microRNA as tumor marker microRNA-126 andmicroRNA-182 are related to urinary bladder cancerrdquo UrologicOncology vol 28 no 6 pp 655ndash661 2010


[89] L Mengual J J Lozano M Ingelmo-Torres C Gazquez MJ Ribal and A Alcaraz ldquoUsing microRNA profiling in urinesamples to develop a non-invasive test for bladder cancerrdquoInternational Journal of Cancer vol 133 no 11 pp 2631ndash26412013

[90] L Xing N W Todd L Yu H Fang and F Jiang ldquoEarlydetection of squamous cell lung cancer in sputum by a panel ofmicroRNAmarkersrdquoModern Pathology vol 23 no 8 pp 1157ndash1164 2010

[91] S M Kim H W Kang W T Kim et al ldquoCell-free microRNA-214 from urine as a biomarker for non-muscle-invasive bladdercancerrdquo Korean Journal of Urology vol 54 no 11 pp 791ndash7962013

[92] X-G Liu W-Y Zhu Y-Y Huang et al ldquoHigh expressionof serum miR-21 and tumor miR-200c associated with poorprognosis in patients with lung cancerrdquo Medical Oncology vol29 no 2 pp 618ndash626 2012

[93] H-B Le W-Y Zhu D-D Chen et al ldquoEvaluation of dynamicchange of serummiR-21 and miR-24 in pre- and post-operativelung carcinoma patientsrdquo Medical Oncology vol 29 no 5 pp3190ndash3197 2012

[94] G-H Liu Z-G Zhou R Chen et al ldquoSerummiR-21 and miR-92a as biomarkers in the diagnosis and prognosis of colorectalcancerrdquo Tumor Biology vol 34 no 4 pp 2175ndash2181 2013

[95] Y Toiyama M Takahashi K Hur et al ldquoSerum miR-21 asa diagnostic and prognostic biomarker in colorectal cancerrdquoJournal of the National Cancer Institute vol 105 no 12 pp 849ndash859 2013

[96] E-H Cai Y-X Gao Z-Z Wei W-Y Chen P Yu and K LildquoSerummiR-21 expression in human esophageal squamous cellcarcinomasrdquo Asian Pacific Journal of Cancer Prevention vol 13no 4 pp 1563ndash1567 2012

[97] P Wang L Zhuang J Zhang et al ldquoThe serum miR-21 levelserves as a predictor for the chemosensitivity of advanced pan-creatic cancer and miR-21 expression confers chemoresistanceby targeting FasLrdquoMolecularOncology vol 7 no 3 pp 334ndash3452013

[98] W Ge D-C Yu Q-G Li X Chen C-Y Zhang and Y-T DingldquoExpression of serum miR-16 let-7f and miR-21 in patientswith hepatocellular carcinoma and their clinical significancesrdquoClinical Laboratory vol 60 no 3 pp 427ndash434 2014

[99] Y-Z Xu Q-H Xi W-L Ge and X-Q Zhang ldquoIdentificationof serum microRNA-21 as a biomarker for early detection andprognosis in human epithelial ovarian cancerrdquo Asian PacificJournal of Cancer Prevention vol 14 no 2 pp 1057ndash1060 2013

[100] C-G Liu G A Calin B Meloon et al ldquoAn oligonucleotidemicrochip for genome-widemicroRNA profiling in human andmouse tissuesrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 101 no 26 pp 9740ndash97442004

[101] M De Planell-Saguer and M C Rodicio ldquoDetection methodsformicroRNAs in clinic practicerdquoClinical Biochemistry vol 46no 10-11 pp 869ndash878 2013

[102] P Mestdagh T Feys N Bernard et al ldquoHigh-throughputstem-loop RT-qPCRmiRNA expression profiling using minuteamounts of input RNArdquo Nucleic Acids Research vol 36 no 21article e143 2008

[103] T D Schmittgen E J Lee J Jiang et al ldquoReal-time PCR quanti-fication of precursor and mature microRNArdquoMethods vol 44no 1 pp 31ndash38 2008

[104] J Shen S A Stass and F Jiang ldquoMicroRNAs as potential bio-markers in human solid tumorsrdquo Cancer Letters vol 329 no 2pp 125ndash136 2013

[105] J R Dijkstra L J M Mekenkamp S Teerenstra I de Krijgerand I D Nagtegaal ldquoMicroRNA expression in formalin-fixedparaffin embedded tissue using real time quantitative PCRthe strengths and pitfallsrdquo Journal of Cellular and MolecularMedicine vol 16 no 4 pp 683ndash690 2012

[106] W Li and K Ruan ldquoMicroRNA detection by microarrayrdquo Ana-lytical and Bioanalytical Chemistry vol 394 no 4 pp 1117ndash11242009

[107] C J Creighton J G Reid and P H Gunaratne ldquoExpressionprofiling of microRNAs by deep sequencingrdquo Briefings in Bio-informatics vol 10 no 5 pp 490ndash497 2009

[108] Y Li Z Zhang F Liu W Vongsangnak Q Jing and B ShenldquoPerformance comparison and evaluation of software toolsfor microRNA deep-sequencing data analysisrdquo Nucleic AcidsResearch vol 40 no 10 pp 4298ndash4305 2012

[109] L Weng X Wu H Gao et al ldquoMicroRNA profiling ofclear cell renal cell carcinoma by whole-genome small RNAdeep sequencing of paired frozen and formalin-fixed paraffin-embedded tissue specimensrdquo Journal of Pathology vol 222 no1 pp 41ndash51 2010

[110] M Wanunu T Dadosh V Ray J Jin L McReynolds and MDrndic ldquoRapid electronic detection of probe-specific microR-NAs using thin nanopore sensorsrdquo Nature Nanotechnology vol5 no 11 pp 807ndash814 2010

[111] Y Wang D Zheng Q Tan M X Wang and L Q GuldquoNanopore-based detection of circulating microRNAs in lungcancer patientsrdquoNature Nanotechnology vol 6 no 10 pp 668ndash674 2010

[112] D Madhavan M Zucknick M Wallwiener et al ldquoCirculatingmiRNAs as surrogate markers for circulating tumor cells andprognostic markers in metastatic breast cancerrdquoClinical CancerResearch vol 18 no 21 pp 5972ndash5982 2012

[113] F Mar-Aguilar J A Mendoza-Ramırez I Malagon-Santiago etal ldquoSerum circulating microRNA profiling for identification ofpotential breast cancer biomarkersrdquo Disease Markers vol 34no 3 pp 163ndash169 2013

[114] X Luo C Stock B Burwinkel and H Brenner ldquoIdentificationand evaluation of plasma microRNAs for early detection ofcolorectal cancerrdquo PLoS ONE vol 8 no 5 Article ID e628802013

[115] A Navarro T Diaz E Gallardo et al ldquoPrognostic implicationsof miR-16 expression levels in resected non-small-cell lungcancerrdquo Journal of Surgical Oncology vol 103 no 5 pp 411ndash4152011

[116] M Boeri C Verri D Conte et al ldquoMicroRNA signaturesin tissues and plasma predict development and prognosis ofcomputed tomography detected lung cancerrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 9 pp 3713ndash3718 2011

[117] Y Ladeiro G Couchy C Balabaud et al ldquoMicroRNA profilingin hepatocellular tumors is associated with clinical features andoncogenetumor suppressor gene mutationsrdquo Hepatology vol47 no 6 pp 1955ndash1963 2008

[118] J F Ji and X W Wang ldquoNew kids on the block diagnostic andprognostic microRNAs in hepatocellular carcinomardquo CancerBiology andTherapy vol 8 no 18 pp 1683ndash1690 2009

[119] A Karakatsanis I Papaconstantinou M Gazouli A Lyber-opoulou G Polymeneas and D Voros ldquoExpression of microR-NAs miR-21 miR-31 miR-122 miR-145 miR-146a miR-200c


miR-221 miR-222 and miR-223 in patients with hepatocellularcarcinoma or intrahepatic cholangiocarcinoma and its prog-nostic significancerdquoMolecular Carcinogenesis vol 52 no 4 pp297ndash303 2013

[120] M D Giraldez J J Lozano G Ramırez et al ldquoCirculatingMicroRNAs as biomarkers of colorectal cancer results from agenome-wide profiling and validation studyrdquo Clinical Gastroen-terology and Hepatology vol 11 no 6 pp 681e3ndash688e3 2013

[121] Q Wang Z Huang S Ni et al ldquoPlasma miR-601 and miR-760 are novel biomarkers for the early detection of colorectalcancerrdquo PLoS ONE vol 7 no 9 Article ID e44398 2012

[122] H Cheng L Zhang D E Cogdell et al ldquoCirculating plasmaMiR-141 is a novel biomarker for metastatic colon cancer andpredicts poor prognosisrdquo PLoS ONE vol 6 no 3 Article IDe17745 2011

[123] G A Ganepola J R Rutledge Suman P Suman A Yieng-pruksawan and D H Chang ldquoNovel blood-based microRNAbiomarker panel for early diagnosis of pancreatic cancerrdquoWorldJournal of Gastrointestinal Oncology vol 6 no 1 pp 22ndash332014

[124] N A Schultz C Dehlendorff B V Jensen et al ldquoMicroRNAbiomarkers in whole blood for detection of pancreatic cancerrdquoJAMAmdashJournal of the American Medical Association vol 311no 4 pp 392ndash404 2014

[125] G Zhao J-G Zhang Y Shi et al ldquoMiR-130b is a prognosticmarker and inhibits cell proliferation and invasion in pancreaticcancer through targeting STAT3rdquo PLoS ONE vol 8 no 9Article ID e73803 2013

[126] W A Mardin and S T Mees ldquoMicroRNAs novel diagnosticand therapeutic tools for pancreatic ductal adenocarcinomardquoAnnals of Surgical Oncology vol 16 no 11 pp 3183ndash3189 2009

[127] Z-H Chen G-L Zhang H-R Li et al ldquoA panel of five circula-ting microRNAs as potential biomarkers for prostate cancerrdquoProstate vol 72 no 13 pp 1443ndash1452 2012

[128] R Mahn L C Heukamp S Rogenhofer A von Ruecker SC Muller and J Ellinger ldquoCirculating microRNAs (miRNA) inserum of patients with prostate cancerrdquo Urology vol 77 no 5pp 1265e9ndash1265e16 2011

[129] J Shen G W Hruby J M McKiernan et al ldquoDysregulationof circulating microRNAs and prediction of aggressive prostatecancerrdquo Prostate vol 72 no 13 pp 1469ndash1477 2012

[130] H-L Zhang L-F Yang Y Zhu et al ldquoSerum miRNA-21elevated levels in patients with metastatic hormone-refractoryprostate cancer and potential predictive factor for the efficacyof docetaxel-based chemotherapyrdquo Prostate vol 71 no 3 pp326ndash331 2011

[131] K E Resnick H Alder J P Hagan D L Richardson CM Croce and D E Cohn ldquoThe detection of differentiallyexpressed microRNAs from the serum of ovarian cancerpatients using a novel real-time PCR platformrdquo GynecologicOncology vol 112 no 1 pp 55ndash59 2009

[132] P P Shah L E Hutchinson and S S Kakar ldquoEmerging roleof microRNAs in diagnosis and treatment of various diseasesincluding ovarian cancerrdquo Journal of Ovarian Research vol 2no 1 article 11 2009

[133] M S Zaman D M Maher S Khan M Jaggi and S CChauhan ldquoCurrent status and implications of microRNAs inovarian cancer diagnosis and therapyrdquo Journal of OvarianResearch vol 5 no 1 article 44 2012

[134] C W S Kan M A Hahn G B Gard et al ldquoElevated levelsof circulating microRNA-200 family members correlate with

serous epithelial ovarian cancerrdquo BMC Cancer vol 12 article627 2012

[135] S Qiu S Lin D Hu Y Feng Y Tan and Y Peng ldquoInteractionsof miR-323miR-326miR-329 and miR-130amiR-155miR-210as prognostic indicators for clinical outcome of glioblastomapatientsrdquo Journal of Translational Medicine vol 11 no 1 article10 2013

[136] A Conti M Aguennouz D La Torre et al ldquomiR-21 and 221upregulation and miR-181b downregulation in human grade II-IV astrocytic tumorsrdquo Journal of Neuro-Oncology vol 93 no 3pp 325ndash332 2009

[137] H F Tu S C Lin and K W Chang ldquoMicroRNA aberrances inhead and neck cancer pathogenetic and clinical significancerdquoCurrent Opinion in Otolaryngology and Head and Neck Surgeryvol 21 no 2 pp 104ndash111 2013

[138] A B Y Hui M Lenarduzzi T Krushel et al ldquoComprehensiveMicroRNA profiling for head and neck squamous cell carcino-masrdquoClinical Cancer Research vol 16 no 4 pp 1129ndash1139 2010

[139] A BHuiW Shi P C Boutros et al ldquoRobust globalmicro-RNAprofiling with formalin-fixed paraffin-embedded breast cancertissuesrdquo Laboratory Investigation vol 89 no 5 pp 597ndash6062009

[140] L F Sempere M Christensen A Silahtaroglu et al ldquoAlteredmicroRNA expression confined to specific epithelial cell sub-populations in breast cancerrdquo Cancer Research vol 67 no 24pp 11612ndash11620 2007

[141] Y-M Lee J-Y Lee C-C Ho et al ldquomiRNA-34b as a tumorsuppressor in estrogen-dependent growth of breast cancercellsrdquo Breast Cancer Research vol 13 no 6 article R116 2011

[142] R Aharonov D Lebanony H Benjamin et al ldquoDiagnosticassay based on hsa-miR-205 expression distinguishes squamousfrom nonsquamous non-small-cell lung carcinomardquo Journal ofClinical Oncology vol 27 no 12 pp 2030ndash2037 2009

[143] F Fornari M Milazzo P Chieco et al ldquoIn hepatocellular car-cinomamiR-519d is up-regulated by p53 and DNA hypomethy-lation and targets CDKN1Ap21 PTEN AKT3 and TIMP2rdquoJournal of Pathology vol 227 no 3 pp 275ndash285 2012

[144] M Rong R He Y Dang and G Chen ldquoExpression andclinicopathological significance of miR-146a in hepatocellularcarcinoma tissuesrdquo Upsala Journal of Medical Sciences vol 119no 1 pp 19ndash24 2014

[145] Q Zhi J Zhu X Guo et al ldquoMetastasis-related miR-185 is apotential prognostic biomarker for hepatocellular carcinoma inearly stagerdquoBiomedicine and Pharmacotherapy vol 67 no 5 pp393ndash398 2013

[146] M Koelz J Lense F Wrba M Scheffler H P Dienes and MOdenthal ldquoDown-regulation of miR-221 and miR-222 corre-lates with pronouncedKit expression in gastrointestinal stromaltumorsrdquo International Journal of Oncology vol 38 no 2 pp503ndash511 2011

[147] M Bloomston W L Frankel F Petrocca et al ldquoMicroRNAexpression patterns to differentiate pancreatic adenocarcinomafrom normal pancreas and chronic pancreatitisrdquo Journal of theAmerican Medical Association vol 297 no 17 pp 1901ndash19082007

[148] T Greither L F Grochola A Udelnow C Lautenschlager PWurl and H Taubert ldquoElevated expression of microRNAs 155203 210 and 222 in pancreatic tumors is associated with poorersurvivalrdquo International Journal of Cancer vol 126 no 1 pp 73ndash80 2010

[149] J Yu K Ohuchida K Mizumoto et al ldquoMicroRNA hsa-miR-200c is an independent prognostic factor in pancreatic cancer


and its upregulation inhibits pancreatic cancer invasion butincreases cell proliferationrdquoMolecular Cancer vol 9 article 1692010

[150] X Hu J K Schwarz J S Lewis Jr et al ldquoA microRNAexpression signature for cervical cancer prognosisrdquo CancerResearch vol 70 no 4 pp 1441ndash1448 2010

[151] A Laios S OToole R Flavin et al ldquoPotential role of miR-9 andmiR-223 in recurrent ovarian cancerrdquo Molecular Cancer vol 7article 35 2008

[152] A Masamune E Nakano S Hamada T Takikawa N Yoshidaand T Shimosegawa ldquoAlteration of the microRNA expressionprofile during the activation of pancreatic stellate cellsrdquo Scandi-navian Journal of Gastroenterology vol 49 no 3 pp 323ndash3312014

[153] S Saini S Majid S Yamamura et al ldquoRegulatory role of mir-203 in prostate cancer progression and metastasisrdquo ClinicalCancer Research vol 17 no 16 pp 5287ndash5298 2011

[154] C W Wu S C Ng Y Dong et al ldquoIdentification of microrna-135b in stool as a potential noninvasive biomarker for colorectalcancer and adenomardquo Clinical Cancer Research vol 20 no 11pp 2994ndash3002 2014

[155] F L Yong C W Law and C W Wang ldquoPotentiality of a triplemicroRNA classifier MiR-193a-3p miR-23a and miR-338-5pfor early detection of colorectal cancerrdquo BMC Cancer vol 13article 280 2013

[156] U Segersten Y Spector Y Goren S Tabak and P-U Malm-strom ldquoThe role of microRNA profiling in prognosticatingprogression in Ta and T1 urinary bladder cancerrdquo UrologicOncology Seminars and Original Investigations vol 32 no 5pp 613ndash618 2014

[157] T Ichimi H Enokida Y Okuno et al ldquoIdentification of novelmicroRNA targets based on microRNA signatures in bladdercancerrdquo International Journal of Cancer vol 125 no 2 pp 345ndash352 2009

[158] C Yang C Wang X Chen et al ldquoIdentification of sevenserum microRNAs from a genome-wide serum microRNAexpression profile as potential noninvasive biomarkers formalignant astrocytomasrdquo International Journal of Cancer vol132 no 1 pp 116ndash127 2013

[159] C Eichelser D Flesch-Janys J Chang-Claude K Pantel andHSchwarzenbach ldquoDeregulated serum concentrations of circu-lating cell-freemicroRNAsmiR-17miR-34amiR-155 andmiR-373 in human breast cancer development and progressionrdquoClinical chemistry vol 59 no 10 pp 1489ndash1496 2013

[160] Q Wu C Wang Z Lu L Guo and Q Ge ldquoAnalysis of serumgenome-wide microRNAs for breast cancer detectionrdquo ClinicaChimica Acta vol 413 no 13-14 pp 1058ndash1065 2012

[161] G Rabinowits C Gercel-Taylor J M Day D D Taylor andG H Kloecker ldquoExosomal microRNA a diagnostic marker forlung cancerrdquoClinical LungCancer vol 10 no 1 pp 42ndash46 2009

[162] P Qi S-Q Cheng H Wang N Li Y-F Chen and C-F Gao ldquoSerum microRNAs as biomarkers for hepatocellularcarcinoma in Chinese patients with chronic hepatitis B virusinfectionrdquo PLoS ONE vol 6 no 12 Article ID e28486 2011

[163] Y Yamamoto N Kosaka M Tanaka et al ldquoMicroRNA-500as a potential diagnostic marker for hepatocellular carcinomardquoBiomarkers vol 14 no 7 pp 529ndash538 2009

[164] J Xu C Wu X Che et al ldquoCirculating MicroRNAs miR-21miR-122 and miR-223 in patients with hepatocellular carci-noma or chronic hepatitisrdquo Molecular Carcinogenesis vol 50no 2 pp 136ndash142 2011

[165] J Qi J Wang H Katayama S Sen and S M Liu ldquoCirculatingmicroRNAs (cmiRNAs) as novel potential biomarkers for hep-atocellular carcinomardquo Neoplasma vol 60 no 2 pp 135ndash1422013

[166] K Z Qu K Zhang H R Li N H Afdhal and M AlbitarldquoCirculating MicroRNAs as biomarkers for hepatocellular car-cinomardquo Journal of Clinical Gastroenterology vol 45 no 4 pp355ndash360 2011

[167] M-Y Song K-F Pan H-J Su et al ldquoIdentification of serummicroRNAs as novel non-invasive biomarkers for early detec-tion of gastric cancerrdquoPLoSONE vol 7 no 3 Article ID e336082012

[168] R Liu C Zhang Z Hu et al ldquoA five-microRNA signatureidentified from genome-wide serum microRNA expressionprofiling serves as a fingerprint for gastric cancer diagnosisrdquoEuropean Journal of Cancer vol 47 no 5 pp 784ndash791 2011

[169] B-S Li Y-L ZhaoGGuo et al ldquoPlasmamicroRNAsmiR-223miR-21 and miR-218 as novel potential biomarkers for gastriccancer detectionrdquo PLoS ONE vol 7 no 7 Article ID e416292012

[170] H Konishi D Ichikawa S Komatsu et al ldquoDetection of gas-tric cancer-associated microRNAs on microRNA microarraycomparing pre- and post-operative plasmardquo British Journal ofCancer vol 106 no 4 pp 740ndash747 2012

[171] J J LaConti N Shivapurkar A Preet et al ldquoTissue and serummicroRNAs in the Kras11986612119863 transgenic animal model and inpatients with pancreatic cancerrdquo PLoS ONE vol 6 no 6 ArticleID e20687 2011

[172] S Yabushita K Fukamachi H Tanaka et al ldquoCirculatingmicrornas in serum of human k-ras oncogene transgenic ratswith pancreatic ductal adenocarcinomasrdquo Pancreas vol 41 no7 pp 1013ndash1018 2012

[173] J Wang J Chen P Chang et al ldquoMicroRNAs in plasma ofpancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of diseaserdquo Cancer Prevention Research vol2 no 9 pp 807ndash813 2009

[174] R Que G Ding J Chen and L Cao ldquoAnalysis of serumexosomalmicroRNAs and clinicopathologic features of patientswith pancreatic adenocarcinomardquo World Journal of SurgicalOncology vol 11 article no 219 2013

[175] L-G Wang and J Gu ldquoSerum microRNA-29a is a promisingnovel marker for early detection of colorectal liver metastasisrdquoCancer Epidemiology vol 36 no 1 pp e61ndashe67 2012

[176] Z Huang D Huang S Ni Z Peng W Sheng and X DuldquoPlasma microRNAs are promising novel biomarkers for earlydetection of colorectal cancerrdquo International Journal of Cancervol 127 no 1 pp 118ndash126 2010

[177] J B Kjersem T Ikdahl O C Lingjaerde T Guren K MTveit and E H Kure ldquoPlasma microRNAs predicting clinicaloutcome in metastatic colorectal cancer patients receiving first-line oxaliplatin-based treatmentrdquo Molecular Oncology vol 8no 1 pp 59ndash67 2014

[178] X-X Pu G-L Huang H-Q Guo et al ldquoCirculating miR-221directly amplified from plasma is a potential diagnostic andprognostic marker of colorectal cancer and is correlated withp53 expressionrdquo Journal of Gastroenterology and Hepatologyvol 25 no 10 pp 1674ndash1680 2010

[179] A E Frampton J Krell T M H Gall L Castellano JStebbing and L R Jiao ldquoMIR-15b and MIR-17 are tumor-derived plasmamicrornas dysregulated in colorectal neoplasiardquoAnnals of Surgery 2014


[180] H Zheng L Zhang Y Zhao et al ldquoPlasma miRNAs asdiagnostic and prognostic biomarkers for ovarian cancerrdquo PLoSONE vol 8 no 11 Article ID e77853 2013

[181] A M Westermann D Schmidt S Holdenrieder et al ldquoSerummicroRNAs as biomarkers in patients undergoing prostatebiopsy results from a prospective multi-center studyrdquo Anti-cancer research vol 34 no 2 pp 665ndash669 2014

[182] F Moltzahn A B Olshen L Baehner et al ldquoMicrofluidic-based multiplex qRT-PCR identifies diagnostic and prognosticmicroRNA signatures in the sera of prostate cancer patientsrdquoCancer Research vol 71 no 2 pp 550ndash560 2011

[183] L Dyrskjoslasht M S Ostenfeld J B Bramsen et al ldquoGenomicprofiling ofmicroRNAs in bladder cancermiR-129 is associatedwith poor outcome and promotes cell death in vitrordquo CancerResearch vol 69 no 11 pp 4851ndash4860 2009

[184] A Tolle M Jung S Rabenhorst E Kilic K Jung and SWeikert ldquoIdentification of microRNAs in blood and urine astumour markers for the detection of urinary bladder cancerrdquoOncology Reports vol 30 no 4 pp 1949ndash1956 2013

[185] Z C Zhang Y Huang X J Wang M Wang and L LMa ldquoExpression of circulating microRNAs in patients withbladder urothelial carcinomardquo Journal of Peking UniversityHealth Sciences vol 45 no 4 pp 532ndash536 2013

[186] G Wang E S-Y Chan B C-H Kwan et al ldquoExpressionof microRNAs in the urine of patients with bladder cancerrdquoClinical Genitourinary Cancer vol 10 no 2 pp 106ndash113 2012

[187] C Fang D-X Zhu H-J Dong et al ldquoSerum microRNAs arepromising novel biomarkers for diffuse large B cell lymphomardquoAnnals of Hematology vol 91 no 4 pp 553ndash559 2012

[188] F Zhi X Cao X Xie et al ldquoIdentification of circulatingMicroRNAs as potential biomarkers for detecting acutemyeloidleukemiardquo PLoS ONE vol 8 no 2 Article ID e56718 2013

[189] B Stamatopoulos N Meuleman B Haibe-Kains et alldquomicroRNA-29c and microRNA-223 down-regulation hasin vivo significance in chronic lymphocytic leukemia andimproves disease risk stratificationrdquo Blood vol 113 no 21 pp5237ndash5245 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of next generation of technologies such as miRNA microar-rays and high-throughput deep sequencing techniques [6]translating biomarker into practice with increased diagnosticand therapeutic sensitivity and specificity would be less of aproblem [7] Among these contexts the potential of miRNAsbeing involved in carcinogenesis as cancer biomarkers hasbeen extensively investigated and developed

In this review we will overview recent advances in thedysregulation of tumor-associated miRNAs in both biopsiesand circulation and highlight some critical challenges for thetransition of miRNAs as cancer biomarkers from a researchsetting into clinical application

2 Overview of miRNAs DefinitionBiogenesis and Functions

miRNAs belong to the heterogeneous class of ncRNAsof 22ndash24 nt RNAs that play important regulatory roles bytargeting mRNAs for cleavage or translational repression [8]By definition miRNAs are small RNA molecules incapableof encoding proteins but possessing important structuralcatalytic and regulatory functions According to the latestmiRBase release (v21 June 2014 httpwwwmirbaseorg)there are 28645 entries representing hairpin precursor miR-NAs from 223 species expressing 35828 mature miRNAproducts MiRNA genes are located either in independentnoncoding DNA loci or in the introns of protein-codinggenes [9] Furthermore up to 50 miRNAs are clusteredon chromosomes with a common promoter [10] and cotran-scribed to form different miRNAs families which sharecommon mRNAs targets and biological processes because oftheir identical seed regions [11]

It is well established that miRNA biogenesis is a com-plex process which generally includes three main steps asfollows (i) In the nucleus miRNA genes are transcriptedby RNA polymerase II to form primary miRNA transcripts(pri-miRNAs) [12] (ii) Subsequently the Drosha RNase IIIendonuclease trims the pri-miRNA to liberate a pre-miRNAhairpin which is actively transported to the cytoplasm byRan-GTP and exportin-5 [8 13] (iii) Its final matura-tion is processed in the cytoplasm where Dicer RNase IIIendonuclease cleaves the pre-miRNA into a single-strandedmature miRNA [13] and then the mature miRNA binds toproteins of the argonaute (Ago) family and assembles theRNA induced silencing complex (RISC) together to exert itsfurther physiological functions

After being incorporated into the RISC the maturemiRNA induces posttranscriptional gene silencing by teth-ering RISC to be partially complementary to the targetmRNA predominantly found within the 31015840-untranslatedregion (UTR) [14] Inspiringly a recent study showed that asingle miRNAmay repress more than 100mRNAs on averageand over 60 of human protein-coding genes are conservedtargets of miRNAs [15] Given this vast majority of mRNAtargets regulated by miRNAs aberrant miRNA expressionprofoundly influences a wide variety of cell regulation path-ways important to cell proliferation [16] apoptosis [17] andstress responses [18] In cancer disease phenotype miRNAs

serve exclusively as a novel class of oncogenes or tumorsuppressor genes named ldquooncomiRsrdquo or ldquotumor suppressivemiRsrdquo [19] Following the first identified tumor suppressivemiRNA genemiR-15amiR-16-1 revolving in the developmentof B-cell lymphocytic leukemia [20] a growing number ofoncomiRs have been validated as well as tumor suppressivemiRs (more details are in the review [21]) Remarkablyit has been continually confirmed by numbers of studiesthat ldquooncomiRsrdquo or ldquotumor suppressive miRsrdquo revolve in amultitude of cancer-related signaling pathways For examplemiR-17 activates PI3KAKT pathway and regulates tumorgrowth [22] the axis of p53-miRNA-34 network regulatesWnt pathway [23] and promotes EMT program [24] andlet-7 leads to growth inhibition by negatively regulatingRAS pathway [25] Taken together these observations givea miRNAs-mediated complex network model that connectsoncogenic and tumor suppressive signaling pathways inhuman cancers indicating that miRNAs may play a moreindispensable role in the pathogenesis of human cancers thanpreviously thought

3 miRNAs as Potential Biomarkers in Cancer

31 Aberrant miRNA Expression Profiles in Cancer

311 Linkage of Aberrant miRNA Expression Profiles withCancer Development Aberrant expression of miRNAs incancer is characterized by abnormal expression levels ofmature or precursor miRNA transcripts in comparison withthose in the corresponding normal tissues A steadily growingnumber of reports have proven that human cancers fre-quently show an aberrant expression profile of miRNAs aswell as pre-miRNAs With various kinds of high-throughputsequencing platforms applied to analyze the genome-wideexpression of miRNA genes these years aberrant expressionprofiles ofmiRNAs either downregulated or upregulated havebeen discovered in a broad variety of human malignant can-cers including lymphoma [26] breast cancer [27] colorectalcancer [28] prostate cancer [29] and glioma [30]

Although the causes of aberrant miRNAs expression incancer have not yet been fully elucidated several definedmechanisms can be possible including chromosomal abnor-malities genomic mutations epigenetic changes and alter-ations in miRNA biogenesis Notably some miRNA genesare frequently located at fragile sites and genomic regionsrelated to cancer [31] In other words alterations of miRNAsexpression can directly reflect the chromosomal or genomicchanges of cancer-associated genes Together with importantroles of cancer-associatedmiRNAs identified in various typesof cancer cell lines [32] and clinical tumor specimens [33ndash35] the aberrant expressions of miRNAs appear to showsignificant values of clinical applications

312 Diagnostic Prognostic and Predictive Values of miRNASignatures for Cancer Given those circumstances abovealterations ofmiRNAs expression in different types of neopla-sia constitute specific patterns ofmiRNA signature for certaintypes of cancer Logically these specific miRNA signatures



















Table1Ty

peso

fspecific

miRNAs

biom

arkersin

different

cancers

Cancer

Type

ofbiom

arker

miRNAs

biom

arkers

Reference

Breastcancer

Diagn

ostic

miR-10b-21-30a-92a-125b-141-145-200-801-155-191-203-210

[112113]

Progno

stic

miR-10b-373miR-210

[4344

]Predictiv

emiR-125b

[53]

Lung

cancer

Diagn

ostic

miR-21-155m

iR-16miR-17-19b-25-29c-30c-106a-126-451-66

0-28-3p

[114ndash116]

Progno

stic

miR-221let-7a-137m

iR-372-182

[4142]

miR-1-15b-16-21-126-142-3p-148a-197

[116]

Predictiv

emiR-128b

[56]

Liverc

ancer

Diagn

ostic

miR-222-223-181a-181b-181c-200c-203-21-224-10b-222126-96

[117118]

Progno

stic

miR-21-22-26-29-31-122-124-135a-139-145-146

a-155-200c-221-222-222-223

[119]

Predictiv

emiR-21-200b

[52]

Colon

cancer

Diagn

ostic

miR-15b-18a-29a-335miR-17-3p-20a-21-92-601-760-29a

[120121]

Progno

stic

miR-141

[122]

Predictiv

emiR-127-3p-92a-486-3p-378let-7

gmiR-181b

[5455]

Pancreaticcancer

Diagn

ostic

miR-205-21m

iR-642b-885-5p-22

[39123]

miR-145-150-223-636-26b-34a-122-126lowast-145-150-223-505-636-8855p

[124]

Progno

stic

miR-130bmiR-21-105-196a-2-203-210-222-452-105-127-187-5

18a-2-3

0a-3p

[125126]

Predictiv

emiR-21

[52]

Prostatecancer

Diagn

ostic

miR-30c-622-1285miR-10b-373let-7

c-7e

[43127]

miR-141-375miR-26a-195

[128]

Progno

stic

miR-141-375miR-20a-21-141-145-221

[45129]

Predictiv

emiR-21

[130]

Ovaria

ncancer

Diagn

ostic

miR-200

familylet-7

familym

iR-21-29a-92-93m

iR-126-127-132-155-214-182-205-144-222-302

[131ndash133]

Progno

stic

miR-410-64

5miR-200

familym

iR-141-429

[46134]

Predictiv


[132]




Glioblastoma








Pancreatic cancer




Prostate cancer























Plasma miR-148b -376c -409-3p -801 Up [80]

Lung cancer




Liver cancer Serum


Gastric cancer





Colorectal cancer


Plasma


Ovarian cancer







Bladder


Urine



Table 3 Continued


























Acknowledgments


References














































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Table1Ty

peso

fspecific

miRNAs

biom

arkersin

different

cancers

Cancer

Type

ofbiom

arker

miRNAs

biom

arkers

Reference

Breastcancer

Diagn

ostic

miR-10b-21-30a-92a-125b-141-145-200-801-155-191-203-210

[112113]

Progno

stic

miR-10b-373miR-210

[4344

]Predictiv

emiR-125b

[53]

Lung

cancer

Diagn

ostic

miR-21-155m

iR-16miR-17-19b-25-29c-30c-106a-126-451-66

0-28-3p

[114ndash116]

Progno

stic

miR-221let-7a-137m

iR-372-182

[4142]

miR-1-15b-16-21-126-142-3p-148a-197

[116]

Predictiv

emiR-128b

[56]

Liverc

ancer

Diagn

ostic

miR-222-223-181a-181b-181c-200c-203-21-224-10b-222126-96

[117118]

Progno

stic

miR-21-22-26-29-31-122-124-135a-139-145-146

a-155-200c-221-222-222-223

[119]

Predictiv

emiR-21-200b

[52]

Colon

cancer

Diagn

ostic

miR-15b-18a-29a-335miR-17-3p-20a-21-92-601-760-29a

[120121]

Progno

stic

miR-141

[122]

Predictiv

emiR-127-3p-92a-486-3p-378let-7

gmiR-181b

[5455]

Pancreaticcancer

Diagn

ostic

miR-205-21m

iR-642b-885-5p-22

[39123]

miR-145-150-223-636-26b-34a-122-126lowast-145-150-223-505-636-8855p

[124]

Progno

stic

miR-130bmiR-21-105-196a-2-203-210-222-452-105-127-187-5

18a-2-3

0a-3p

[125126]

Predictiv

emiR-21

[52]

Prostatecancer

Diagn

ostic

miR-30c-622-1285miR-10b-373let-7

c-7e

[43127]

miR-141-375miR-26a-195

[128]

Progno

stic

miR-141-375miR-20a-21-141-145-221

[45129]

Predictiv

emiR-21

[130]

Ovaria

ncancer

Diagn

ostic

miR-200

familylet-7

familym

iR-21-29a-92-93m

iR-126-127-132-155-214-182-205-144-222-302

[131ndash133]

Progno

stic

miR-410-64

5miR-200

familym

iR-141-429

[46134]

Predictiv


[132]




Glioblastoma








Pancreatic cancer




Prostate cancer























Plasma miR-148b -376c -409-3p -801 Up [80]

Lung cancer




Liver cancer Serum


Gastric cancer





Colorectal cancer


Plasma


Ovarian cancer







Bladder


Urine



Table 3 Continued


























Acknowledgments


References














































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Glioblastoma








Pancreatic cancer




Prostate cancer























Plasma miR-148b -376c -409-3p -801 Up [80]

Lung cancer




Liver cancer Serum


Gastric cancer





Colorectal cancer


Plasma


Ovarian cancer







Bladder


Urine



Table 3 Continued


























Acknowledgments


References














































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Plasma miR-148b -376c -409-3p -801 Up [80]

Lung cancer




Liver cancer Serum


Gastric cancer





Colorectal cancer


Plasma


Ovarian cancer







Bladder


Urine



Table 3 Continued


























Acknowledgments


References














































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 3 Continued


























Acknowledgments


References














































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Acknowledgments


References














































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article micrornas as potential biomarkers...

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