journal.pone.0002557

Aberrant Expression of Oncogenic and Tumor-Suppressive MicroRNAs in Cervical Cancer Is Required for Cancer Cell Growth

Xiaohong Wang1.,Shuang Tang1.,Shu-Yun Le2,Robert Lu1,Janet S.Rader3,Craig Meyers4,Zhi-Ming Zheng1*

1HIV and AIDS Malignancy Branch,Center for Cancer Research,Nation Cancer Institute(NCI)/National Institutes of Health(NIH),Bethesda,Maryland,United States of America,2Nanobiology Program,Center for Cancer Research,Nation Cancer Institute(NCI)/National Institutes of Health(NIH),Bethesda,Maryland,United States of America,3Department of Obstetrics and Gynecology,Washington University School of Medicine,St.Louis,Missouri,United States of America,4Department of Microbiology and Immunology,Penn State University College of Medicine,Hershey,Pennsylvania,United States of America

Abstract

MicroRNAs(miRNAs)play important roles in cancer development.By cloning and sequencing of a HPV16+CaSki cell small RNA library,we isolated174miRNAs(including the novel miR-193c)which could be grouped into46different miRNA species,with miR-21,miR-24,miR-27a,and miR-205being most abundant.We chose for further study10miRNAs according to their cloning frequency and associated their levels in10cervical cancer-or cervical intraepithelial neoplasia-derived cell lines.No correlation was observed between their expression with the presence or absence of an integrated or episomal HPV genome.All cell lines examined contained no detectable miR-143and miR-145.HPV-infected cell lines expressed a different set of miRNAs when grown in organotypic raft cultured as compared to monolayer cell culture,including expression of miR-143and miR-145.This suggests a correlation between miRNA expression and tissue https://www.wendangku.net/doc/852924805.html,ing miRNA array analyses for age-matched normal cervix and cervical cancer tissues,in combination with northern blot verification,we identified significantly deregulated miRNAs in cervical cancer tissues,with miR-126,miR-143,and miR-145downregulation and miR-15b,miR-16,miR-146a,and miR-155upregulation.Functional studies showed that both miR-143and miR-145are suppressive to cell growth.When introduced into cell lines,miR-146a was found to promote cell proliferation.Collectively, our data indicate that downregulation of miR-143and miR-145and upregulation of miR-146a play a role in cervical carcinogenesis.

Citation:Wang X,Tang S,Le S-Y,Lu R,Rader JS,et al.(2008)Aberrant Expression of Oncogenic and Tumor-Suppressive MicroRNAs in Cervical Cancer Is Required for Cancer Cell Growth.PLoS ONE3(7):e2557.doi:10.1371/journal.pone.0002557

Editor:Dong-Yan Jin,University of Hong Kong,China

Received April10,2008;Accepted May13,2008;Published July2,2008

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that,once placed in the public domain,this work may be freely reproduced,distributed,transmitted,modified,built upon,or otherwise used by anyone for any lawful purpose.

Funding:This research was supported by the Intramural Research Program of the NIH,National Cancer Institute,Center for Cancer Research and by NIH grants CA94141and CA95713to J.S.R.and CA79006to C.M.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:zhengt@https://www.wendangku.net/doc/852924805.html,

.These authors contributed equally to this work.

Introduction

Cervical cancer is one of the most common cancers in women worldwide,with an estimated global incidence of470,000new cases and approximately233,000deaths per year[1,2].Cervical cancer is the leading cause of death from cancer in many low-resource countries where widespread screening by cervical cytology is unavailable.The incidence is lower in developed countries as a consequence of cervical screening and of ongoing active health education programs.Cervical cancer rates in the United States were estimated as10,370new cases and3,710 deaths in2006[3].The causal relationship between high-risk HPV(HR-HPV)infection and cervical cancer has been well documented in epidemiological and functional studies.High-risk HPVs,such as HPV16,HPV18,and HPV31,have been detected in up to99.7%of cervical squamous cell carcinomas and94%–100%of cervical adeno-and adenosquamous carcinomas[4,5]. The high-risk HPV oncoproteins,E6and E7,contribute to cervical carcinogenesis by inactivating the cellular tumor suppres-sor proteins p53and pRb,respectively[6–8].

miRNAs are regulatory,non-coding RNAs about21–23 nucleotides in length and are expressed at specific stages of tissue development or cell differentiation,and have large-scale effects on the expression of a variety of genes at the post-transcriptional level. Through base-pairing with its targeted mRNAs,a miRNA induces RNA degradation or translational suppression of the targeted transcripts[9,10].Cellular miRNAs are transcribed by RNA polymerase II as long,capped,polyadenylated primary miRNA (pri-miRNA)transcripts bearing a stem-loop hairpin structure of ,80-nts[11].Mature miRNAs result from the processing of pri-miRNAs in two sequential cleavage steps mediated by two RNase III enzymes,Drosha and Dicer[12].Drosha,in complex with DGCR8,cleaves the pri-miRNA at a specified distance(approx-imately11bp)from the stem–single stranded RNA junction in the nucleus to give a pre-miRNA of an,60-nt hairpin with a2-nt39 overhang[13,14].After the pre-miRNA is exported to the

cytoplasm by exportin5[15,16],it is then recognized by Dicer,in complex with dsRBD-containing partner HIV-1TAR RNA-binding protein(TRBP)[17],to remove the terminal loop,leaving a second2-nt39overhang[18].The resulting21-to23-nt miRNA products contain a2-nt39overhang at each strand and act as the functional intermediates of RNAi that direct mRNA cleavage and translational attenuation.Although their biological functions remain largely unknown,recent studies suggest that miRNAs contribute to the development of various cancers[19]and might function as an important component of the cell’s natural defense against viral infection[20–22].It has been proposed that an unique miRNA expression profile for a particular cancer would be a useful biomarker for cancer diagnosis[23]and prognosis[24].In this study,we used various approaches to profile miRNA expression from cervical cancer tissues and cervical cancer–derived cell lines as well as from HPV-infected vaginal keratinocytes.We demonstrate that a subset of miRNAs is significantly dysregulated in cervical cancers and pre-neoplastic lesions.

Results

miRNA expression profile in cervical cancer cell lines

To investigate the expression profiles of miRNAs in cervical cancer cells,we isolated total cell RNA from cervical cancer–derived CaSki C-2cells,which contain,500copies of the HPV16 genome per cell.The fractionated RNAs with sizes of15–30nts were cloned and sequenced based on a published protocol[25]. Overall,we cloned a total of174miRNAs from363cDNA clones which were categorized into46different miRNA species(Table1), with miR-21,miR-24,miR-27a,and miR-205being most abundant.No HPV16-derived miRNA was cloned in this screening,all the miRNAs were cellular.Interestingly,we found that some copies of the cloned miR-21and miR-205had heterogenous39ends,with an extra C nucleotide on the39end of68%of the miR-21copies and an extra U on the39end of33% of the miR-205copies,presumably derived from wobble digestion of the pre-miRNAs by Dicer(Fig.1).In addition,a new subspecies of miR-193,miR-193c(GenBank accession number:EF100863), was identified three times from the screening and was verified by northern blotting.The miR-193c subspecies differs from miR-

193b by one nucleotide at position nt21and by the lack of three Us at the39end(miR-193b,59-aacuggcccucaaagucccg c uuu-39; miR-193c,59-aacuggcccucaaagucccg a-39).The profile of the newly identified miR-193c which is3-nt smaller than miR-193b was similar to that of miR-21in HeLa cells and293cells,but less abundant than miR-27a.HeLa and293cells produced no detectable miR-205as a doublet as seen in CaSki cells(Fig.2A). Based on these cloning results,we further screened several available cervical cancer cell lines with or without HPV infection by northern blotting for the expression of8miRNAs with relatively high cloning frequency and2miRNAs(miR-143and miR-145)which were not isolated in our cloning(Table1).Eight cervical cancer cell lines;two isolates of the W12cell line(20861 with integrated HPV16genomes and20863with episomal HPV16genomes)originally isolated from a cervical intraepithelial neoplasia(CIN)biopsy tissues[26]and HaCaT cells(an immortalized HPV-negative skin keratinocyte line)[27]were examined for the expression of the10miRNAs.A paired total RNAs from cervical cancer and adjacent normal cervical tissues commercially obtained from Ambion were also compared.All cell lines and cervical cancer tissues showed a similar miRNA expression profile.This miRNA profile differed from normal cervical tissues with reduced expression of miR-27a,miR-143,and miR-145(Fig.2B).In contrast,the cervical cancer tissue and the cell lines,except SiHa(HPV16+),HeLa(HPV18+),and C33A cells (HPV2),had increased expression of miR-205when compared with normal cervical tissue(Fig.2B).SiHa,HeLa,and C33A cells have no expression of miR-205.In all of the cell lines,expression of the miR-143and-145was less than what was observed in the cervical cancer tissue.Despite the presence of some differences in the expression of individual miRNA from one cell line to another, we were unable to specify an expression profile of those detected miRNAs in correlation to the presence of integrated or episomal HPV genomes.The downregulation of miR-143,which is derived from the same miRNA precursor of miR-145[28],was further confirmed to be cancer-specific by comparing its expression in cervical cancer tissues to normal cervix(Fig.2C).

miRNA expression profile in normal and cancerous cervical tissues

Since our cloning approach has a limitation in the isolation of low abundant miRNAs and northern blotting is limited by the probe chosen,the two methods may not be the best to distinguish a difference from one cell line to another in miRNA expression profile.To further investigate whether miRNAs are differentially expressed in cervical cancer tissues,we collected five pairs of age-matched normal and cancerous cervical tissues and compared Table1.miRNA expression profile in HPV16-positive CaSki C-2cells by cDNA cloning.

miRNA

names

Frequency of

detection*miRNA names

Frequency of

detection*

miR-71miR-921

miR-15a2miR-932

miR-167miR-961

miR-17-5p8miR-106b1

miR-19a1miR-130a2

miR-19b1miR-130b1

miR-203miR-135b1

miR-2131miR-1413

miR-222miR-1511

miR-23a5miR-1862

miR-2410miR-193b1

miR-251miR-193c3

miR-26b1miR-2032

miR-27a29miR-20512

miR-27b8miR-3012

miR-29a3miR-4242

miR-29b1let-7a6

miR-30a-3p1let-7b2

miR-30a-5p2let-7d2

miR-30d1let-7e1

miR-313let-7f1

miR-331let-7i1

miR-34a1

miR-34c1Total174

*Isolation frequency from363cDNA clones.CaSki C-2cells stably express a hairpin-derived HPV16E7siRNA with no interfering function on HPV16E7 expression(72).This screening also isolated3copies of the E7siRNA.

doi:10.1371/journal.pone.0002557.t001

their expression profiles using a miRNA array analysis containing 455miRNAs.Only four sample pairs qualified for the final clustering analysis as determined by sample consistency analyses.An expression abundance analysis,based on a signal density $20,000,showed that both normal and cancerous cervical tissues had abundant expression of miR-23a,miR-23b,let-7a,let-7c,and let-7d,whereas high expression of miR-26a,miR-29a,miR-99a,miR-100,miR-125b,miR-143,miR-145,miR195,and miR-199a was only observed in normal cervical tissues,and high expression of miR-16,miR-21,miR-205,and let-7f was only observed in cervical cancer tissues,despite that relatively low levels of miR-16and miR-21were noticed from a homogeneous cell population in

some cell lines (Fig.2B).Expression of miR-143and miR-145showed more than 2.7-fold reduction in the cervical cancer https://www.wendangku.net/doc/852924805.html,ing a clustering analysis,we observed an significant increased expression of 18miRNAs in cervical cancer and 15miRNAs in normal cervix (Fig.3A,miRNAs in red color)(P ,0.05,t-test).For verification,a set of selected miRNA probes were used to perform a northern blot analysis for two normal and two cancer samples.We were able to confirm that the cancer tissues had reduced expression of miR-126and miR-424,and increased expression of miR-15b,miR-16,miR-146a,miR-155,and miR-223(Fig.3B and 3C),after individual miRNA level in each sample was quantified and normalized to U6expression.

miRNA expression profile in HPV-infected,keratinocyte-derived raft tissues

To investigate whether the observed miRNA signatures in cervical cancer tissues are specific to cervical cancer,we examined miRNA expression in pre-neoplastic lesions.The lack of conven-tional cell cultures for HPV multiplication has largely restrained our understanding of the HPV life cycle and pathogenesis.However,infectious HPVs have been successfully produced from raft cultures derived from human keratinocytes [29,30]and induction of pre-neoplastic lesions in raft tissues by HPV infection with the morphology similar to that of cervical intraepithelial neoplasia [31]makes this system of great advantage for our https://www.wendangku.net/doc/852924805.html,ing miRNA array analyses,we compared the expression profiles of 455human miRNAs in HPV18-infected primary human vaginal keratinocytes (HVKs)in monolayer cultures and in stratified and

differentiated raft tissues.An expression abundance analysis,based on a signal density $20,000,showed that miR-21,miR-23a,miR-23b,miR-26a,miR-205,let-7c,and let-7f abundantly expressed in HVKs both in monolayer cultures and raft cultures.Three miRNAs abundantly expressed only in monolayer cultures (miR-24,miR-29a,and miR-221)and 11miRNAs abundantly expressed only in the stratified and differentiated raft cultures (miR-27a,miR-27b,miR-200b,miR-200c,miR-203,miR-638,let-7a,let-7b,let-7d,let-7e,and let-7g).Using a clustering analysis,we further determined that 16miRNAs were upregulated (Fig.4A,raft in red)and 25miRNAs were downregulated (Fig.4A,raft in green)in the raft cultures when compared to the expression levels in the correspond-ing monolayer cultures (P ,0.05,t-test).To confirm the observa-tions,the paired samples were analyzed for miRNA expression by northern blotting using a set of specific miRNA probes (Fig.4B and 4C).Quantitation of each miRNA level in each sample was normalized to U6expression.We confirmed that the expression of miR-26b,miR-195,and miR-200c was increased in the raft cultures,but the expression of miR-143and miR-145was decreased (Fig.4B and 4C).The increased abundance of miR-200c showing 67%more expression in rafts than in monolayers (P ,0.08)by array analysis was verified by northern blotting.Although upregulation of miR-15a and miR-223and downregulation of miR-218and miR-424were observed both in the rafts (pre-neoplastic lesions)and in cervical cancer tissues,the majority of the miRNA expression signatures showed no correlation between the two tissues (compare Fig.4A to Fig.3A).However,downregulation of miR-146a was noticed in the raft cultures rather than the upregulation seen in cervical cancer tissues and was further confirmed by northern blotting (Fig.5),indicating that the upregulation of miR-146a expression is cervical cancer-specific.In contrast,a slight increased expression of miR-21and miR-26b was observed in both the pre-neoplastic lesions (HPV-infected rafts)and cervical cancer tissues and thus their expression appears not cancer-specific (Fig.5).

Reduced miR-143and miR-145in cervical lesions and cervical cancer are suppressive in cervical cancer cell growth

Having demonstrated a significant downregulation of miR-143and miR-145in cervical cancer and pre-neoplastic lesions,we

Figure 1.Wobble digestion of miRNAs by Dicer in CaSki cells.Arrows above the pre-miRNA indicate wobble-digestion sites on the pre–miR-21in A and pre-miR-205in B ,producing products with additional nucleotides on the 39end.The sequences shown in red correspond to mature miRNA while that shown in black is the portion of pre-miRNA which is removed during processing.doi:10.1371/journal.pone.0002557.g001

investigated whether this downregulation is important in develop-ment of cervical cancer.To address this question,synthetic miR-143and miR-145precursors were transiently transfected into HeLa cells and the effect of overexpression of their mature miRNAs on HeLa cell growth was evaluated by cell counting.As shown in Fig.6,both miR-143and miR-145were found suppressive (p ,0.005for miR-143and p ,0.008of miR-145,t -test)to HeLa cell growth.This suppressive effect on the cell growth was not an immediate cell response;rather,two consecutive cell transfections with each miRNA at an interval of 48hrs were needed.Data suggest that both miR-143and miR-145probably need to be downregulated in cervical cells for tumor

progression.

Figure 2.miRNA expression profile in cervical cancer cells determined by northern blot analysis.A .miR-193c expression in CaSki,HeLa,and 293cells.CaSki and its subclones C-V and C-2cells [72]were compared with HeLa (HPV18+)and 293cells (HPV 2)for expression of miR-193c by northern blotting.Total RNA (30m g)from CaSki,HeLa,and 293cells was separated in a 15%denaturing polyacrylamide gel,blotted onto a Gene Screen-Plus membrane,and then probed separately with a sense (s)or an antisense (as)miR-193c probe.Subsequently,the membrane was reprobed with antisense probes for miR-21,miR-27a,miR-205,or U6,respectively.B .miRNA expression in cervical cancer–derived cell lines.Total cell RNA (30m g)was extracted from various cell lines with or without HPV infection.Northern blotting was used to examine the expression of 10miRNAs from eight cancer cell lines,two W12cell subclones,HaCaT cells,normal cervix,and cervical cancer tissues (Ambion).C .Downregulation of miR-143in cervical cancer tissues.Total RNAs from two pairs of normal (NT)cervix vs cervical cancer (Ca)tissues from clinical samples and one pair of normal cervix and cervical cancer purchased from Ambion were compared for the expression of miR-143by northern blotting.U6was also blotted as an internal control for sample loading in panel A ,B ,and C .doi:10.1371/journal.pone.0002557.g002

Increased miR-146a in cervical cancer promotes cell proliferation

Considering a6-fold increase of miR-146a expression in cervical cancer tissues,we hypothesized that a high level of miR-146a might play a role in cervical cancer cell growth.Our initial approach using anti-miR-146a inhibitors or using a morphonino-oligo to block miR-146a maturation to knock down miR-146a expression in cervical cancer cell lines failed to induce a phenotypic effect(data not shown).To our surprise,we found that all cervical cancer cell lines examined expressed no detectable miR-146a(Fig.7A).Subsequently,miR-146a was introduced by transient transfection into HeLa cells,a HPV18+cervical cancer cell line,and HCT116cells,a colorectal cancer cell line(Fig.7B).

A slow,but significantly enhanced cell proliferation of both HeLa (p,0.009,t-test)and HCT116(P,0.019,t-test)cells was noticed in the presence of exogenous miR-146a.By calculation of the cell counts from each group,we demonstrated that both HeLa cells and HCT116cells containing miR-146a had a doubling time2–3hrs faster than that of the cells with miR-controls(Fig.7C and 7D).This data suggests that miR-146a functions as a growth factor in cervical cancer.

Discussion

In this study,we demonstrated miRNA expression profiles in cervical cancer and HPV infection-induced pre-neoplastic lesions in raft tissues.Although both cervical cancer tissues and HPV-infected raft tissues with pre-neoplastic lesions showed an upregulation of miR-15a and miR-223and downregulation of miR-143,miR-145,miR-218,and miR-424,the majority of the miRNA expression showed no correlation between the two tissues and might delineate the disease progression.However,a high level of miR-146a expression was found to be cervical cancer-specific since its expression was down-regulated both in normal tissues and in HPV-infected raft tissues with pre-neoplastic lesions.

Figure3.miRNA signatures in cervical cancer and normal cervical tissues by miRNA array analysis.Total RNA(5m g)isolated from age-matched normal cervical tissues(NT)and cervical cancer tissues(Ca)were analyzed by miRNA array analysis.The cancer tissue1,3,and5were infected with HPV16and the cancer tissue4was infected with HPV45.A.A clustering graph was created using a hierarchical method from those miRNAs with signal density of more than1000and shows increased(red)or decreased(green)expression(P,0.05)of individual miRNA from4age-matched normal and cancerous cervical tissues(Table S1).The column heading indicates individual sample code in the assay.Each row shows the expression pattern of individual miRNA as a log2-transformed expression ratio,with the most closely related expression joined by a branch and clustered by an average-linked algorithm.Branch lengths reflect degrees of similarity between miRNA expression patterns.Color scale on the top of the panel represents the degree of expression based on a calibrated ratio.Green indicates lower expression compared to the mean(zero),black indicates expression equal to the mean,and red indicates higher expression compared to the mean.B.Verification of miRNA expression profile by northern blotting.Total cell RNA(30m g)isolated from each tissue was probed with individual antisense oligo to each indicated miRNA.An additional band in each blot indicates a wobble product from Dicer digestion.C.Bar graphs show the relative levels of the examined miRNAs normalized to U6 RNA in each tissue in panel B.

doi:10.1371/journal.pone.0002557.g003

Finding of downregulation of miR-143and miR-145and upregulation of miR-146a in cervical cancer was in particular attractive for two reasons:(1)miR-143and miR-145are expressed from the same miRNA precursor [28]and are downregulated also in HPV-induced pre-neoplastic lesions,in our case in HPV-infected raft tissues,suggesting that their reduction take place in an early step before cancer development.Downregulation of miR-143and miR-145has been found in several other cancers,including colorectal cancer [32],B-cell lymphoma [33],and recently in cervical cancer [34,35].Thus,our finding is important for understanding the mechanisms by which miR-143and miR-145are involved in carcinogenesis.Inhibition by miR-143and miR-145of HeLa cell growth implies that miR-143and miR-145probably need to be

downregulated in order for cancer growth.(2)Upregulation of miR-146a in cervical cancer tissues,but not in HPV-induced pre-neoplastic lesions or in cancer-derived cell lines indicates that miR-146expression is cervical cancer-specific.Various studies show that miR-146a is a NF-kappaB-dependent gene [36–38].Expression of miR-146a appears to vary in different cancer tissues in which an increased level of miR-146a was observed in Burkitts’lymphoma lines with EBV-LMP1(latent membrane protein 1)expression [37,38],but a decreased level in hormone-refractory prostate cancer [39]and papillary thyroid carcinoma [40].In cervical cancer,upregulation of miR146a expression appears unrelated to any of HPV gene expression since its expression was even reduced in productively HPV-infected raft tissues.Interestingly,all

cervical

Figure 4.miRNA expression from undifferentiated keratinocytes in monolayers and stratified and differentiated keratinocytes in raft tissues.Raft tissues (cultures)with HPV18infection were derived from human vaginal keratinocytes (HVKs)in monolayer cultures with HPV18DNA transfection.Total cell RNA (5m g each)extracted from each type of cells was compared in parallel for miRNA expression using miRNA array analyses.A .A clustering graph was created by a hierarchical method from those miRNAs with a signal density of more than 1000obtained from three independent transfections in each group (Table S2).The graph shows decreased (green)or increased (red)expression (p ,0.05)of individual miRNAs from undifferentiated HVKs in monolayers (Mono)versus stratified and differentiated HVKs in rafts.See other details in Fig.3A.B .Verification of miRNA expression from undifferentiated HVKs in monolayers and stratified and differentiated HVKs in rafts by northern blotting.Total cell RNA (30m g each)isolated from differentiated or undifferentiated HVKs was probed with selected miRNA probes.C .Bar graphs show the relative levels of the examined miRNAs normalized to U6RNA in each sample in panel B .M =monolayer culture,R =raft.doi:10.1371/journal.pone.0002557.g004

cancer cell lines and a skin keratinocyte line examined contains no detectable miR-146a,suggesting that miR-146a in cervical cancer tissues is upregulated by a factor missing in a homogenous cell population or is expressed by another type of cells in cancer tissues.Thus,we conclude that upregulation of miR-146a expression in cervical cancer is beneficial for cancer growth as introduction of miR-146a into cancer cells increased cell doubling time and promoted cell proliferation.

Approximately 800human miRNAs have been predicted.Of these,more than 450(https://www.wendangku.net/doc/852924805.html,/sequences,2006)have now been identified in the human [41,42][43,44],and their functions are beginning to be elucidated [45].In this study,we cloned and identified 174miRNAs which were grouped into 46different miRNA species.A new subspecies of miR-193,miR-193c,was also cloned and identified from our miRNA library screening.Screening of 10cell lines derived from cervical cancer or CIN tissues for expression of 10miRNAs suggested that the presence or absence of integrated or episomal HPV genomes has no effect on the expression of analyzed miRNAs.Moreover,we were unable to identify a single HPV16-derived miRNA from HPV16+CaSki cells by this approach,although other nuclear DNA viruses do encode miRNAs [46],including EBV [47];KSHV [48–50];HSV-1[51,52];and SV40[53].A negative cloning for HPV16-derived miRNAs suggests that the integrated HPV16genome in CaSki cells appears not to express viral miRNAs.Another study using differentiated HPV31+LKP1cells containing ,50copies of an episomal HPV31genome per cell also failed to identify virus-derived miRNAs [54].

Recent reports have suggested that miRNAs contribute to a variety of cell functions [41]and are involved in the development of human cancers [19].For example,miR-32controls cell resistance to a retroviral infection [55].A cluster of six miRNAs on human chromosome 13is regulated by c-Myc,and the c-Myc–regulated miR-17-5p and miR-20a negatively modulate the expression of E2F1,a transcription factor [56].miRNAs have been also characterized recently as potential oncogenes that promote the

development of human B-cell lymphoma (miR-17-92cluster)[57]and the proliferation and tumorigenesis of primary human cells (miR-372and miR-373)by neutralizing p53-mediated CDK inhibition [58].Another study suggested that overexpression of miR-17-5p,miR-20a,miR-21,miR-92,miR-106a,and miR-155could be considered an miRNA signature of solid cancer [59].In this report,18miRNAs were upregulated and 15miRNAs were downregulated in cervical cancer tissues.The increased expression of miR-15b,miR-16,miR-146a,miR-155,and miR-223that we observed in cervical cancer tissues has also been implicated in the development of other human cancers:miR-15and miR-16regulate apoptosis by targeting BCL2[60]and their mutation has been associated with chronic lymphocytic leukemia [61];miR-16is also involved in control of cytokine RNA instability [62];miR-146b levels are highly increased in papillary thyroid carcinoma [40];miR-155has recently been implicated in the development of lymphoblastic leukemia/high-grade lymphoma [63]and lung cancer [24]and in the regulation of human fibroblast angiotensin II type 1receptor expression [64];miR-223,along with the transcription factors C/EBPa and NFI-A,participates in regulation of granulocytic differentiation by suppressing the transcription of NFI-A mRNA [65].

Although mature miRNAs derived from their primary tran-scripts through Drosha and Dicer digestion in two separate trimming reactions contain a 2-nt 39overhang on each strand,Dicer digestion of the pre-miRNA is not always performed precisely.This was reflected in the miR-21and miR-205isolated in this study.We found that more than 68%of the miR-21isolated from CaSki cells had an additional C at the 39end and 33%of the miR-205isolated contained an extra U at the 39end,but no extra nucleotide was ever identified from the 59ends of either miRNA.When compared to the corresponding pre-miRNA sequence,the additional C on the miR-21and the U on the miR-205were found to be derived directly from the nucleotide 39to the cleavage site,suggesting that Dicer digestion has a wobble mechanism.This observation by small RNA cloning can be further verified as double bands for miR-21(Fig.5A and Fig.7A)and for

miR-205

Figure 5.Upregulation of miR-146a is cervical cancer-specific.A .Total RNAs from HVKs in two rafts and two monolayer (Mono)cultures were compared with two cervical cancer (Ca)tissues for the expression of miR-146a by northern blotting.The expression levels of miR-21and miR-26b were examined as miRNA controls.Small nuclear RNA U6was also blotted as an internal control for sample loading.B .Bar graphs show the relative levels of the examined miRNAs normalized to U6RNA in each sample in panel A .M =monolayer culture,R =raft.doi:10.1371/journal.pone.0002557.g005

(Fig.2A and 2B)in northern blot analyses.Our findings are consistent with other reports obtained from biochemical analyses that Dicer uses a 39end counting rule and measures a distance of ,22nt from the 39-terminus to cleave both strands of RNA,with 1-to 2-nt shifts in the preferred cleavage position [18,66].

Materials and Methods Cells,tissues,and raft cultures

Eight cervical cancer cell lines were used for the assays:HPV16+CaSki and SiHa,HPV18+HeLa and C4II,HPV68+ME180[67],

and HPV31+CIN6126E and 9E cells.Two HPV16+W12subclones (20861and 20863)originally isolated from a low-grade cervical lesion histologically diagnosed as CIN I [26]were also used for comparison.Among these cell lines,20863and 9E cells contain an episomal form of the HPV genome,and 20861and 6E cells have an integrated HPV genome [68,69].A HPV-negative cervical cancer cell line,C33A [70],and a HPV-negative human keratinocyte line,HaCaT [27],were also included in this study.All cell lines,except for W12,6E,and 9E cells,were grown in Dulbecco’s modified Eagle’s medium (DMEM)with 10%FBS at 37u C and 5%CO 2.The W12subclones and CIN6126E and 9E were grown on pretreated NIH3T3feeder cells [71].Human primary vaginal keratinocytes and their derived raft cultures were prepared as described [30].The stratified and differentiated keratinocytes in the raft culture epidermal layer were collected free from collagen (no fibroblasts)and frozen on dry ice for total cell RNA preparation.HCT116cell line,a colorectal cancer cell line,was a gift from Dr.Bert Vogelstein of Johns Hopkins University and grown in McCoy’s 5A medium with 10%FBS at 37u C and 5%CO 2.Total cell RNA was prepared from each cell line using TRIzol Reagent (Invitrogen,Carlsbad,CA).

Age-matched normal cervix and cervical cancer tissues were obtained from anonymized excess cervical tissues that were no longer needed for diagnostic or clinical https://www.wendangku.net/doc/852924805.html,e of these tissues was approved both by the Washington University Medical Center Human Studies Committee and by the NIH Office of Human Subjects Research.Each tissue was homogenized in an Eppedorf tube in 1ml TRIzol Reagent using an electric homogenizer (Omni International,Marietta,GA)with a separate disposible probe for each tissue.The isolated RNA was dissolved in RNase-free water and stored in a 270u C freezer.

miRNA cloning from cervical cancer cells

Cloning of miRNAs from cervical cancer cells was performed as described previously.Briefly,approximately 600m g of total cell RNA prepared from HPV16-positive CaSki C-2cells [a CaSki subclone cell line [72]was dissolved in RNA loading buffer II (Ambion,TX)and denatured at 75u C for 5min.The RNA samples were then fractionated,along with 32P-labeled RNA oligos as size markers,in a 15%denaturing polyacrylamide gel containing 8M urea.After the gel was exposed to an X-ray film,the small RNAs corresponding to the size range of the 15-nt and 30-nt oligos were cut from the gel,eluted with elution buffer (0.3M NaCl,0.5%SDS),and incubated overnight at 37u C.Subsequently,the small RNAs were ethanol precipitated and dephosphorylated with alkaline phosphatase and then ligated to a 59-phosphorylated,32P-labeled 39adapter (59-rUrUrUAACCGC-GAATTCCAG-L,L represents 39end protection with C7-amino modifiers,rU represents ribonucleotide)with T4RNA ligase.After ligation,the ligated products were separated from unligated 39adapters in a 15%denaturing polyacrylamide gel and eluted overnight at 37u C from the gel slices with the elution buffer described above.After ethanol precipitation,the recovered small RNAs were phosphorylated with T4polynucleotide kinase and ligated with a 59adapter (59-ACGGAATTCCTCACTrArArA-39,rA represents ribonucleotide)in the presence of T4RNA ligase.The ligated RNAs were then separated from unligated 59adapters in a 15%denaturing polyacrylamide gel and eluted from the gel slices as described above.Reverse transcription was then conducted with a reverse primer (59-GACTAGCTG-GAATTCGCGGTTAAA-39)and Superscript II (Invitrogen).The cDNA was amplified with the first PCR primer (59-CAGCCAACGGAATTCCTCACTAAA-39)and the reverse primer for 20cycles of 45sec at 94u C,85sec at 50u C,

and

Figure 6.Overexpression of miR-143and miR-145suppresses HeLa cell growth.A .miR-143and miR-145are suppressive for HeLa cell growth.Arrows indicate the time points when the cells received a specific miRNA (30nM)transfection.Viable cells in each group were counted at the indicated time points.Data represent the mean 6SD of two experiments,each performed in triplicate.B .Relative levels of miR-143and miR-145in HeLa cells at 96hrs after first transient transfection.Total cell RNA extracted was examined by miRNA ligation assay.A tRNA level in each sample was used as a loading control.doi:10.1371/journal.pone.0002557.g006

60sec at 72u C.The PCR products were reamplified with a second pair of PCR primers (59-GACTAGCTTGGTGCCGAATTC-GCGGTTAAA-39,59-GAGCCAACAGGCACCGAATTCCT CACTAAA-39)for 10cycles in the same conditions.The second PCR products were digested with Ban I,concatamerized with T4DNA ligase,and cloned into a pCR2.1-TOPO vector.The bacterial colonies were first screened with PCR using two M13primers (59-GTAAAACGACGGCCAG-39;59-CAGGAAA-CAGCTATGAC-39).Colonies containing an insert from 400bp to1000bp in size were further grown and each plasmid DNA prepared was sequenced from both directions with M13primers.

m Paraflo TM miRNA microarray assay

A microarray assay was performed using a service provider (LC Sciences,Houston,TX).The assay started with 5m g of total RNA sample.The RNA was size-fractionated using a YM-100Microcon centrifugal filter (GE Millipore,Billerica,MA),and the 39ends of the small RNAs (,300nt)were extended with a poly(A)tail using poly(A)polymerase.An oligonucleotide tag was then ligated to the poly(A)tail for later fluorescent dye staining;two different tags were used for the two RNA samples in dual-sample experiments.Hybridization was performed overnight on a m Paraflo microfluidic

chip using a microcirculation pump [73,74].On the microfluidic chip,each detection probe consisted of a chemically modified nucleotide coding segment complementary to the target miRNA (455human miRNA [hsa-miRNA]entries in version 8.1from miRBase,https://www.wendangku.net/doc/852924805.html,/sequences/)or other con-trol RNAs and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate.The detection probes were made by in situ synthesis using photogenerated reagent chemistry.The hybridization melting temperatures were balanced by chemical modifications of the detection probes.Hybridization was performed in 100m L of 66SSPE buffer (0.90M NaCl,60mM Na 2HPO 4,6mM EDTA,pH 6.8)containing 25%formamide at 34u C.After hybridization,the miRNAs were detected by fluorescence labeling using tag-specific Cy3and Cy5dyes.Hybridization images were collected using a laser scanner (GenePix 4000B,Molecular Device)and digitized using Array-Pro image analysis software (Media Cybernetics).Data were analyzed by first subtracting the back-ground and then normalizing the signals using a locally-weighted regression filter [75].For two-color experiments,the ratio of the two sets of detected signals (log2transformed,balanced)was calculated,and the significance of the differences were determined by t -test analysis;significantly different signals were those with p values

less

Figure 7.Overexpression of miR-146a promotes cell proliferation.A .No expression of miR-146a in cervical cancer-derived cell lines and in a human skin-derived keratinocyte line,HaCaT cells.Total cell RNA was extracted from individual cell line and examined for miR-146a and miR-21expression by northern blotting.U6in each sample served as sample loading control.B .Relative level of miR-146a in HeLa and HCT116cells at 96hrs after the first transfection.Total cell RNA extracted from each group was examined by northern blotting.C and D .miR-146a promotes cell proliferation.Arrows indicate the time points when the cells received transfection with miR-146a or miR-control (30nM).Viable cells in each group were counted at the indicated time points.Data represent the mean 6SD of two experiments,each performed in triplicate.doi:10.1371/journal.pone.0002557.g007

than0.05.Multi-array normalization and clustering analysis were performed using a hierarchical method,with an average linkage and Euclidean distance metric.The clustering plots were generated using MultiExperiment Viewer software(v4.0,2006)from The Institute for Genomic Research and were created from those miRNAs with a total signal density of more than1000. Northern blot of small RNAs

Total cell RNA(30m g)was resolved in a12%or15%(Fig.2 only)denaturing polyacrylamide gel,transferred onto a GeneSc-reen-Plus Hybridization Transfer membrane(NEN PerkinElmer, Waltham,MA)in0.56TBE,and blotted with a32P-labeled miRNA-specific oligo as described[72].After blotting,the same membrane was stripped in0.16SSPE,0.5%SDS at85u C to 90u C for20min and prehybridized with hybridization buffer (Perfecthyb,Sigma)for2h and then hybridized with another probe overnight at40u C.The membrane was washed in26SSPE containing0.5%SDS,0.56SSPE containing0.5%SDS,and 0.26SSPE containing0.1%SDS for30min each at40u C,and then exposed to a PhosphorImager screen.The image was captured using a Molecular Dynamic PhosphorImager Storm860 and analyzed with ImageQuant software.The oligo probes were designed based on individual miRNA sequence information deposited in miRBase(https://www.wendangku.net/doc/852924805.html,).An anti-sense oligo of U6snRNA(oST197,59-AAAATATG-GAACGCTTCACGA-39)was used to detect U6snRNA from each sample as a loading control.

miRNA ligation assay

miRNA ligation assay was performed using miRtect-IT TM miRNA Labeling and Detection Kit(USB,Cleveland,OH). Briefly,a detection oligo was59end labeled with c-32P-ATP and OptiKinase.The miRNA and the radiolabeled detection oligo were captured with a miRNA-specific bridge oligo and were then ligated with T4DNA ligase.The ligated miRNA was separated on a12%denaturing polyacrylamide gel and exposed to a PhosphorImager screen for2.5h.The image was captured and analyzed as described above.Transient miRNA transfection

Approximately 2.36105HeLa and HCT116cells were transfected with30nM of individual testing miRNA precursor or control-miRNA precursor(non-specific)purchased from Ambion.In each miRNA transfection,cell passage and miRNA transfection were conducted simultaneously using siPORT TM NeoFX TM Transfection Agent(Ambion)according to the manufacturer’s instruction.After48h,the cells were counted, split,and transfected again with each corresponding miRNA with the same dose.After additional48h,the cells were counted and total RNA was extracted with TRIzol Reagent(Invitrogen).A student t-test was used for statistical analysis of cell counts. Supporting Information

Table S1

Found at:doi:10.1371/journal.pone.0002557.s001(0.08MB XLS)

Table S2

Found at:doi:10.1371/journal.pone.0002557.s002(0.06MB XLS)

Acknowledgments

We thank Jeanette Gullett for her technical assistance,Ernest Kawasaki and David Peterson for their production assistance of our initial microRNA arrays,Carl Baker,Alison McBride,Paul Lambert,and Bert Vogelstein for providing cell lines,David Goldstein for his office support,and Douglas Lowy and Robert Yarchoan for their comments and suggestions in the course of the study and critical reading of our manuscript.Part of this work was presented at the23th International Papillomavirus Conference and Clinical Workshop in Prague,Czech Republic,September1–7,2006and Keystone Symposium on MicroRNA and Cancer,Keystone,Colorado, June8–12,2007.

Author Contributions

Conceived and designed the experiments:ZZ.Performed the experiments: XW ST.Analyzed the data:ZZ SL CM XW ST RL JR.Contributed reagents/materials/analysis tools:CM JR.Wrote the paper:ZZ CM XW JR.

References

1.Parkin DM,Bray F,Ferlay J,Pisani P(2001)Estimating the world cancer

burden:Globocan2000.Int J Cancer94:153–156.

2.Bosch FX,de Sanjose S(2003)Human papillomavirus and cervical cancer–

burden and assessment of causality.J Natl Cancer Inst Monogr(31):3–13. 3.Jemal A,Siegel R,Ward E,Murray T,Xu J,et al.(2006)Cancer statistics,2006.

CA Cancer J Clin56:106–130.

4.Walboomers JM,Jacobs MV,Manos MM,Bosch FX,Kummer JA,et al.(1999)

Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol189:12–19.

5.Castellsague X,Diaz M,de Sanjose S,Munoz N,Herrero R,et al.(2006)

Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors:implications for screening and prevention.J Natl Cancer Inst98: 303–315.

6.Scheffner M,Werness BA,Huibregtse JM,Levine AJ,Howley PM(1990)The

E6oncoprotein encoded by human papillomavirus types16and18promotes the degradation of p53.Cell63:1129–1136.

7.Dyson N,Howley PM,Munger K,Harlow E(1989)The human papilloma

virus-16E7oncoprotein is able to bind to the retinoblastoma gene product.

Science243:934–937.

8.Boyer SN,Wazer DE,Band V(1996)E7protein of human papilloma virus-16

induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway.Cancer Res56:4620–4624.

9.Lewis BP,Burge CB,Bartel DP(2005)Conserved seed pairing,often flanked by

adenosines,indicates that thousands of human genes are microRNA targets.Cell 120:15–20.

10.Farh KK,Grimson A,Jan C,Lewis BP,Johnston WK,et al.(2005)The

widespread impact of mammalian MicroRNAs on mRNA repression and evolution.Science310:1817–1821.

11.Lee Y,Kim M,Han J,Yeom KH,Lee S,et al.(2004)MicroRNA genes are

transcribed by RNA polymerase II.EMBO J23:4051–4060.12.Cullen BR(2004)Transcription and processing of human microRNA

precursors.Mol Cell16:861–865.

13.Lee Y,Ahn C,Han J,Choi H,Kim J,et al.(2003)The nuclear RNase III

Drosha initiates microRNA processing.Nature425:415–419.

14.Han J,Lee Y,Yeom KH,Nam JW,Heo I,et al.(2006)Molecular basis for the

recognition of primary microRNAs by the Drosha-DGCR8complex.Cell125: 887–901.

15.Yi R,Qin Y,Macara IG,Cullen BR(2003)Exportin-5mediates the nuclear

export of pre-microRNAs and short hairpin RNAs.Genes Dev17:3011–3016.

16.Lund E,Guttinger S,Calado A,Dahlberg JE,Kutay U(2004)Nuclear export of

microRNA precursors.Science303:95–98.

17.Chendrimada TP,Gregory RI,Kumaraswamy E,Norman J,Cooch N,et al.

(2005)TRBP recruits the Dicer complex to Ago2for microRNA processing and gene silencing.Nature436:740–744.

18.Zhang H,Kolb FA,Jaskiewicz L,Westhof E,Filipowicz W(2004)Single

processing center models for human Dicer and bacterial RNase III.Cell118: 57–68.

19.Calin GA,Croce CM(2006)MicroRNA signatures in human cancers.Nat Rev

Cancer6:857–866.

20.Fritz JH,Girardin SE,Philpott DJ(2006)Innate immune defense through RNA

interference.Sci STKE(339):pe27.

21.Cullen BR(2006)Is RNA interference involved in intrinsic antiviral immunity in

mammals?Nat Immunol7:563–567.

22.Obbard DJ,Jiggins FM,Halligan DL,Little TJ(2006)Natural selection drives

extremely rapid evolution in antiviral RNAi genes.Curr Biol16:580–585. 23.Lu J,Getz G,Miska EA,Alvarez-Saavedra E,Lamb J,et al.(2005)MicroRNA

expression profiles classify human cancers.Nature435:834–838.

24.Yanaihara N,Caplen N,Bowman E,Seike M,Kumamoto K,et al.(2006)

Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.

Cancer Cell9:189–198.

25.Pfeffer S,Lagos-Quintana M,Tuschl T(2003)Cloning of small RNA molecules.

In:Ausubel FM,Brent R,Kingston RE,Moore DD,Seidman JG,et al.(2003) Current Protocols in Molecular Biology.New York:John Wiley&Sons,Inc.pp

26.4.1–26.4.18.

26.Stanley MA,Browne HM,Appleby M,Minson AC(1989)Properties of a non-

tumorigenic human cervical keratinocyte cell line.Int J Cancer43:672–676.

27.Boukamp P,Petrussevska RT,Breitkreutz D,Hornung J,Markham A,et al.

(1988)Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol106:761–771.

https://www.wendangku.net/doc/852924805.html,ndgraf P,Rusu M,Sheridan R,Sewer A,Iovino N,et al.(2007)A

mammalian microRNA expression atlas based on small RNA library sequencing.Cell129:1401–1414.

29.McLaughlin-Drubin ME,Christensen ND,Meyers C(2004)Propagation,

infection,and neutralization of authentic HPV16virus.Virology322:213–219.

30.McLaughlin-Drubin ME,Meyers C(2005)Propagation of infectious,high-risk

HPV in organotypic‘‘raft’’culture.Methods Mol Med119:171–186.

31.Lowy DR,Howley PM(2001)Papillomaviruses.In:Knipe DM,Howley PM,

Griffin DE,Lamb RA,Martin MA,et al.(2001)Fields Virology.Philadelphia: Lippincott Williams&Wilkins.pp2231–2264.

32.Slaby O,Svoboda M,Fabian P,Smerdova T,Knoflickova D,et al.(2007)

Altered expression of miR-21,miR-31,miR-143and miR-145is related to clinicopathologic features of colorectal cancer.Oncology72:397–402.

33.Akao Y,Nakagawa Y,Kitade Y,Kinoshita T,Naoe T(2007)Downregulation of

microRNAs-143and-145in B-cell malignancies.Cancer Sci98:1914–1920.

34.Lui WO,Pourmand N,Patterson BK,Fire A(2007)Patterns of known and

novel small RNAs in human cervical cancer.Cancer Res67:6031–6043. 35.Martinez I,Gardiner AS,Board KF,Monzon FA,Edwards RP,et al.(2008)

Human papillomavirus type16reduces the expression of microRNA-218in cervical carcinoma cells.Oncogene27:2575–2582.

36.Taganov KD,Boldin MP,Chang KJ,Baltimore D(2006)NF-kappaB-

dependent induction of microRNA miR-146,an inhibitor targeted to signaling proteins of innate immune responses.Proc Natl Acad Sci U S A103: 12481–12486.

37.Cameron JE,Yin Q,Fewell C,Lacey M,McBride J,et al.(2008)Epstein-Barr

virus latent membrane protein1induces cellular MicroRNA miR-146a,a modulator of lymphocyte signaling pathways.J Virol82:1946–1958.

38.Motsch N,Pfuhl T,Mrazek J,Barth S,Grasser FA(2007)Epstein-Barr Virus-

Encoded Latent Membrane Protein1(LMP1)Induces the Expression of the Cellular MicroRNA miR-146a.RNA Biol4:131–137.

39.Lin SL,Chiang A,Chang D,Ying SY(2008)Loss of mir-146a function in

hormone-refractory prostate cancer.RNA14:417–424.

40.He H,Jazdzewski K,Li W,Liyanarachchi S,Nagy R,et al.(2005)The role of

microRNA genes in papillary thyroid carcinoma.Proc Natl Acad Sci U S A102: 19075–19080.

41.Shivdasani RA(2006)MicroRNAs:regulators of gene expression and cell

differentiation.Blood108:3646–3653.

42.Bentwich I,Avniel A,Karov Y,Aharonov R,Gilad S,et al.(2005)Identification

of hundreds of conserved and nonconserved human microRNAs.Nat Genet37: 766–770.

43.Berezikov E,Guryev V,van de BJ,Wienholds E,Plasterk RH,et al.(2005)

Phylogenetic shadowing and computational identification of human microRNA genes.Cell120:21–24.

44.Kim VN,Nam JW(2006)Genomics of microRNA.Trends Genet22:165–173.

45.Krutzfeldt J,Poy MN,Stoffel M(2006)Strategies to determine the biological

function of microRNAs.Nat Genet38Suppl:S14–S19.

46.Cullen BR(2006)Viruses and microRNAs.Nat Genet38Suppl:S25–S30.

47.Pfeffer S,Zavolan M,Grasser FA,Chien M,Russo JJ,et al.(2004)Identification

of virus-encoded microRNAs.Science304:734–736.

48.Pfeffer S,Sewer A,Lagos-Quintana M,Sheridan R,Sander C,et al.(2005)

Identification of microRNAs of the herpesvirus family.Nat Methods2:269–276.

49.Cai X,Lu S,Zhang Z,Gonzalez CM,Damania B,et al.(2005)Kaposi’s

sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells.Proc Natl Acad Sci U S A102:5570–5575.

50.Samols MA,Hu J,Skalsky RL,Renne R(2005)Cloning and identification of a

microRNA cluster within the latency-associated region of Kaposi’s sarcoma-associated herpesvirus.J Virol79:9301–9305.

51.Gupta A,Gartner JJ,Sethupathy P,Hatzigeorgiou AG,Fraser NW(2006)Anti-

apoptotic function of a microRNA encoded by the HSV-1latency-associated transcript.Nature442:82–85.52.Cui C,Griffiths A,Li G,Silva LM,Kramer MF,et al.(2006)Prediction and

identification of herpes simplex virus1-encoded microRNAs.J Virol80: 5499–5508.

53.Sullivan CS,Grundhoff AT,Tevethia S,Pipas JM,Ganem D(2005)SV40-

encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells.Nature435:682–686.

54.Cai X,Li G,Laimins LA,Cullen BR(2006)Human papillomavirus genotype31

does not express detectable microRNA levels during latent or productive virus replication.J Virol80:10890–10893.

55.Lecellier CH,Dunoyer P,Arar K,Lehmann-Che J,Eyquem S,et al.(2005)A

cellular microRNA mediates antiviral defense in human cells.Science308: 557–560.

56.O’Donnell KA,Wentzel EA,Zeller KI,Dang CV,Mendell JT(2005)c-Myc-

regulated microRNAs modulate E2F1expression.Nature435:839–843.

57.He L,Thomson JM,Hemann MT,Hernando-Monge E,Mu D,et al.(2005)A

microRNA polycistron as a potential human oncogene.Nature435:828–833.

58.Voorhoeve PM,le Sage C,Schrier M,Gillis AJ,Stoop H,et al.(2006)A genetic

screen implicates miRNA-372and miRNA-373as oncogenes in testicular germ cell tumors.Cell124:1169–1181.

59.Volinia S,Calin GA,Liu CG,Ambs S,Cimmino A,et al.(2006)A microRNA

expression signature of human solid tumors defines cancer gene targets.Proc Natl Acad Sci U S A103:2257–2261.

60.Cimmino A,Calin GA,Fabbri M,Iorio MV,Ferracin M,et al.(2005)miR-15

and miR-16induce apoptosis by targeting BCL2.Proc Natl Acad Sci U S A102: 13944–13949.

61.Calin GA,Ferracin M,Cimmino A,Di Leva G,Shimizu M,et al.(2005)A

MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia.N Engl J Med353:1793–1801.

62.Jing Q,Huang S,Guth S,Zarubin T,Motoyama A,et al.(2005)Involvement of

microRNA in AU-rich element-mediated mRNA instability.Cell120:623–634.

63.Costinean S,Zanesi N,Pekarsky Y,Tili E,Volinia S,et al.(2006)Pre-B cell

proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155transgenic mice.Proc Natl Acad Sci U S A103:7024–7029.

64.Martin MM,Lee EJ,Buckenberger JA,Schmittgen TD,Elton TS(2006)

MicroRNA-155regulates human angiotensin II type1receptor expression in fibroblasts.J Biol Chem281:18277–18284.

65.Fazi F,Rosa A,Fatica A,Gelmetti V,De Marchis ML,et al.(2005)A

minicircuitry comprised of microRNA-223and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis.Cell123:819–831.

66.Vermeulen A,Behlen L,Reynolds A,Wolfson A,Marshall WS,et al.(2005)The

contributions of dsRNA structure to Dicer specificity and efficiency.RNA11: 674–682.

67.Longuet M,Beaudenon S,Orth G(1996)Two novel genital human

papillomavirus(HPV)types,HPV68and HPV70,related to the potentially oncogenic HPV39.J Clin Microbiol34:738–744.

68.Jeon S,Lambert PF(1995)Integration of human papillomavirus type16DNA

into the human genome leads to increased stability of E6and E7mRNAs: implications for cervical carcinogenesis.Proc Natl Acad Sci U S A92: 1654–1658.

69.Hummel M,Hudson JB,Laimins LA(1992)Differentiation-induced and

constitutive transcription of human papillomavirus type31b in cell lines containing viral episomes.J Virol66:6070–6080.

70.Crook T,Wrede D,Vousden KH(1991)p53point mutation in HPV negative

human cervical carcinoma cell lines.Oncogene6:873–875.

71.Jeon S,Allen-Hoffmann BL,Lambert PF(1995)Integration of human

papillomavirus type16into the human genome correlates with a selective growth advantage of cells.J Virol69:2989–2997.

72.Tang S,Tao M,McCoy JP,Zheng ZM(2006)Short-term induction and long-

term suppression of HPV16oncogene silencing by RNA interference in cervical cancer cells.Oncogene25:2094–2104.

73.Gao X,Gulari E,Zhou X(2004)In situ synthesis of oligonucleotide microarrays.

Biopolymers73:579–596.

74.Zhou X,Cai S,Hong A,You Q,Yu P,et al.(2004)Microfluidic PicoArray

synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences.Nucleic Acids Res32:5409–5417.

75.Bolstad BM,Irizarry RA,Astrand M,Speed TP(2003)A comparison of

normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics19:185–193.