分子生物学知识拓展 (6).pdf

RESEARCHOpen AccessLoss of miR-638 in vitro promotes cell invasionand a mesenchymal-like transition by influencingSOX2 expression in colorectal carcinoma cellsKelong Ma1,2,3,Xiaorong Pan1,Pingsheng Fan4,Yinghua He1,Jun Gu1,Wei Wang1,Tengyue Zhang4,Zonghai Li1*and Xiaoying Luo1*AbstractBackground:Colorectal carcinoma(CRC)is a major cause of cancer mortality.The aberrant expression of severalmicroRNAs is associated with CRC progression;however,the molecular mechanisms underlying this phenomenonare unclear.Methods:miR-638 and SRY-box 2(SOX2)expression levels were detected in 36 tumor samples and their adjacent,non-tumor tissues from patients with CRC,as well as in 4 CRC cell lines,using real-time quantitative RT-PCR(qRT-PCR).SOX2 expression levels were detected in 90 tumor samples and their adjacent tissue using immunohistochemistry.Luciferase reporter and Western blot assays were used to validate SOX2 as a target gene of miR-638.The regulationof SOX2 expression by miR-638 was assessed using qRT-PCR and Western blot assays,and the effects of exogenousmiR-638 and SOX2 on cell invasion and migration were evaluated in vitro using the HCT-116 and SW1116 CRC cell lines.Results:We found that miR-638 expression was differentially impaired in CRC specimens and dependent on tumorgrade.The inhibition of miR-638 by an antagomiR promoted cell invasion and a mesenchymal-like transition(lamellipodium stretching increased and cell-cell contacts decreased,which was accompanied by the suppression ofthe epithelial cell marker ZO-1/E-cadherin and the upregulation of the mesenchymal cell marker vimentin).A reporterassay revealed that miR-638 repressed the luciferase activity of a reporter gene coupled to the 3-untranslated regionof SOX2.miR-638 overexpression downregulated SOX2 expression,and miR-638 inhibition upregulated SOX2 expression.Moreover,miR-638 expression levels were correlated inversely with SOX2 mRNA levels in human CRC tissues.TheRNAi-mediated knockdown of SOX2 phenocopied the invasion-inhibiting effect of miR-638;furthermore,SOX2overexpression blocked the miR-638-induced CRC cell transition to epithelial-like cells.Conclusions:These results demonstrate that the loss of miR-638 promotes invasion and a mesenchymal-like transitionby directly targeting SOX2 in vitro.These findings define miR-638 as a new,invasion-associated tumor suppressor of CRC.Keywords:miR-638,SOX2,CRC,InvasionBackgroundMicroRNAs(miRNAs)play pivotal roles in physiologicaland pathological processes via their regulation of a widevariety of genes,predominantly through their interactionwith the 3-untranslated regions(3UTR)of their corre-sponding mRNA targets 1,2.More than 4,665 maturemiRNA products have been annotated in the humangenome,according to the most recent version of themiRBase program(Release 20:June 2013;http:/www.mirbase.org/),and increasing evidence has shown thatthe deregulation of miRNAs is involved in the pathogen-esis of a wide range of diseases,such as human cancers3,4.However,the roles of most miRNAs in tumor initi-ation and progression are still unknown.Colorectal carcinoma(CRC)is the fourth-most com-mon cause of cancer-related mortality worldwide 5.Approximately 715,000 deaths from CRC are estimated*Correspondence:;Equal contributors1State Key Laboratory of Oncogenes&Related Genes,Shanghai CancerInstitute,Renji Hospital,Shanghai Jiaotong University School of Medicine,No.25/Ln2200,XieTu Rd,Shanghai 200032,ChinaFull list of author information is available at the end of the article 2014 Ma et al.;licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the CreativeCommons Attribution License(http:/creativecommons.org/licenses/by/2.0),which permits unrestricted use,distribution,andreproduction in any medium,provided the original work is properly credited.The Creative Commons Public DomainDedication waiver(http:/creativecommons.org/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.Ma et al.Molecular Cancer 2014,13:118http:/www.molecular- occur annually,accounting for 8%of all cancer deaths6.Because of advancements in CRC treatment regi-mens,there has been substantial progress in the treat-ment for colorectal cancer,and survival rates haveimproved over the past 40 years 7.Metastasis is themajor concern in cancer therapy;cell invasion and theepithelial-to-mesenchymal transition(EMT)is the pri-mary step in this process.The EMT is a biological process in which a polarizedepithelial cell,which normally interacts with the base-ment membrane via its basal surface,undergoes multiplebiochemical changes that cause the epithelial cell to as-sume a mesenchymal cell phenotype.These phenotypicchanges include enhanced migratory capacity,invasiveness,elevated resistance to apoptosis,and a greatly increasedproduction of ECM components 8.Mounting evidencesuggests that the EMT occurs in CRC 9,10.Recent stud-ies have revealed that miRNAs are involved in the EMTprocess in CRC cells;for example,miR-101 11,miR-21212,miR-155 13,miR-130b 14,and miR-34 15 havebeen found to be involved in the EMT process in CRCcells.One study has provided evidence that miR-638 isdownregulated at the invasive front of CRC 16;however,its expression and function were not addressed.In the present study,we sought to determine the roleof miR-638 in CRC progression.We defined miR-638 asa new,invasion-associated tumor suppressor miRNAin vitro.Moreover,we identified SOX2,a factor that caninduce pluripotent stem cells 17,as a direct,functionaltarget of miR-638.Materials and methodsPatients and tissue microarrayParticipants who provided samples also provided writ-ten,informed consent to participate in this study.TheEthics Committee of the Shanghai Cancer Institute ap-proved the study,the consent procedure,and the tissuearray study.All of the research was performed in China.Paired colorectal tumor tissues and their correspondingadjacent non-tumor colorectal tissues(5 cm away fromthe lesions)were collected from patients who underwentcurative surgery for CRC at Anhui Medical University,Anhui Province,China.Normal colon tissue was col-lected from patients with non-cancerous colon disease.A CRC diagnosis was confirmed by histological examin-ation,and the relevant clinical and pathological informa-tion was retrieved from the hospital database(Additionalfile 1:Table S1a).Glass-slide tissue arrays for CRC werepurchased from the Shanghai Outdo Biotech Co.,Ltd.(Shanghai,China)(Additional file 1:Table S2a),and immu-nostaining(SOX2,ab75485,1:100,Abcam,Cambridge,MA;vimentin,#5741,1:50,Cell Signaling Technology,Beverly,MA)was performed on the tissue microarrayslides.Staining was analyzed based on the percentageof positively stained cells and staining intensity by apathologist or using Image-Pro Plus 6.0 software(MediaCybernetics,Inc.,Bethesda,MD)(Additional file 2).Cell cultureFour human CRC cell lines were purchased from theAmerican Type Culture Collection(ATCC,Rockville,MD,USA).HCT-116 cells(ATCC No.CCL-247)weremaintained in McCoys 5A medium,LoVo cells(ATCCNo.CCL-229)were maintained in F-12 K medium(Kaighns Modification of Hams F-12 Medium),andSW480 cells(ATCC No.CCL-228)and SW1116 cells(ATCC No.CCL-233)were maintained in Leibovitzs L-15medium.The media were supplemented with 10%fetalbovine serum,and the cells were incubated in a humidi-fied atmosphere of 95%air and 5%CO2at 37C.TransfectionsmiRNA mimics and miRNA antagomiRs were designedand synthesized by RiboBio(Guangzhou,China).ThemiRNA antagomiRs were composed of nucleotides witha 2-O-methylmodification.The SOX2 siRNAs(sense,5-GGAAUGGACCUUGUAUAGAUC-3;anti-sense,5-UCUAUACAAGGUCCAUUCCCC-3)were synthesizedby RiboBio(Guangzhou,China),and the SOX2 overex-pression construct was obtained from Origene Inc.(Beijing,China).The miRNA mimics,miRNA antagomiRs,SOX2-targeted siRNA,and SOX2 overexpression con-struct were transiently co-transfected with GFP(trans-fection efficiency control)using an Amaxa Nucleofector(Amaxa,Koeln,Germany)according to the manufacturersprotocol.RNA extraction and quantitative real-time RT-PCR(qRT-PCR)Total RNA was extracted using TRIzol reagent(Invitrogen,Carlsbad,CA)according to the manufacturers protocol.qRT-PCR with miRNA was performed using the TaqManReverse Transcription Kit(Applied Biosystems),TaqManMicroRNA Assays(Applied Biosystems),and a Light-Cycler TaqMan Master(Roche Diagnostics,Mannheim,Germany)according to the manufacturers instructions.The miRNA expression levels were calculated using thedelta-delta Ct method with RNU6B as an internal control.A Ct value of 35 was set as the cut-off value for defining asnon-detected.cDNA was reverse-transcribed from 1 g of RNAusing the SYBRPrime Script RT-PCR kit(TakaraBiochemicals,Tokyo,Japan),and the reactions wereperformed on an ABI PRISM7900HT Real-Time PCRSystem.The thermal cycling conditions were as follows:an initial step at 95C for 15 s followed by 40 cycles of95C for 5 s and 60C for 30 s.Each experiment was per-formed in a 20-l reaction volume containing 10 l ofSYBR Prime Ex Taq II(2),0.8 l of forward primer andMa et al.Molecular Cancer 2014,13:118Page 2 of 13http:/www.molecular- primer(10 M each),0.4 l of ROX Reference Dyeor Dye II(50),2 l of cDNA,and 6 l of H2O.-Actin was used as an internal control.The quantifica-tion of the mRNA was calculated using the comparative Ct(the threshold cycle)method according to the followingformula:Ratio=2ct=2 Ct(sample)Ct(calibrator),whereCt is equal to the Ct of the target gene minus the Ctof the endogenous control gene(-actin).The primersfor SOX2(SOX2RTF:5-CGAGATAAACATGGCAATCAAAAT-3;SOX2RTR:5-AATTCAGCAAGAAGCCTCTCCTT-3)have been described previously 18.The in-ternal control actin primers were designed as previouslydescribed 19,20.Western blot analysisProteins were separated by SDS-PAGE and transferred to apolyvinylidene fluoride membrane(Bio-Rad,Hercules,CA).The membrane was blocked with 5%non-fat milk and in-cubated with the following antibodies:the epithelial cellmarker ZO-1(1:200)and E-cadherin(1:1000),the mesen-chymal cell marker vimentin(1:50,000),SOX2(1:2,000),Myc(1:2,000),-actin(1:2,000,Santa Cruz Biotechnology,Santa Cruz,CA),and GAPDH(1:10,000;Kang-ChenBio-tech Shanghai,China).And the quantification ofWestern Blot exerted by ImagineJ software(NIH,USA).Cell migration and invasion assaysWe transfected the miR-638 mimics and SOX2 siRNAinto HCT-116 and SW1116 cells using an AmaxaNucleofector(Amaxa,Koeln,Germany).Cell migrationand invasion were evaluated using Matrigel-uncoatedand-coated transwell chambers(cat.3422,Corning,NY).Briefly,5104cells were suspended in 200 l of DMEMwithout serum and placed into cell culture inserts(8-mpore size;BD Falcon,San Jose,CA)of a companion plate(BD Falcon San Jose,CA)in pre-warmed culture mediumcontaining 10%fetal bovine serum in the well.The cellswere incubated overnight at 37C in 5%CO2and thenfixed in 4%paraformaldehyde in PBS.Cell migration andinvasion were determined by staining the cells with 0.1%crystal violet(Sigma,St Louis,MO)and counting the cellsunder a light microscope(100 magnification)in eightrandomly selected areas.Luciferase reporter assayLuciferase activity was detected using the Dual LuciferaseAssay(Promega,USA)according to the manufacturersprotocols.The transfected cells were lysed in tissue culturedishes with lysis buffer,and the lysates were centrifuged atmaximum speed for 1 min in an Eppendorf microcentri-fuge.The relative luciferase activity was determined usinga Modulus TD20/20 Luminometer(Turner Biosystems,Sunnyvale,CA),and the transfection efficiency was nor-malized to Renilla activity.Immunofluorescence imagingTransfected SW1116 cells were seeded at a density of2104onto poly-L-lysine-coated glass coverslips in a 6-well plate.After further culture overnight,the cells werepermeabilized with 0.1%Triton X-100(Sigma-Aldrich,St.Louis,MO).For filamentous actin(F-actin)staining,the coverslips were incubated with TRITC-labeled phal-loidin(Sigma-Aldrich,St.Louis,MO)at room tem-perature,and the cell nuclei were counterstained withDAPI.The cells were co-transfected with 40 ng of pEGFPplasmid as a control.Statistical analysesAll experiments were performed in triplicate.The dataare presented as the mean values standard error of themean(SEM)and were analyzed using Students t-test.p values less than 0.05 were considered significant.Statis-tical analyses were performed using GraphPad Prism 5.01software(GraphPad Software Inc.,San Diego,CA).The accession numbers for miR-638 is MIMAT0003308,and that for SOX2 is NM_003106.2.ResultsmiR-638 shows reduced expression in colorectal carcinomaPrevious microarray analyses revealed that 23 miRNAsare downregulated in CRC tissues(Additional file 1:Table S3),including miR-497 21,miR-9 22,miR-30a23,and miR-139 24.To further screen miRNAs thatare deregulated in CRC,qRT-PCR assays were conductedto evaluate the expression levels of these miRNAs in 36pairs of CRC clinical samples.In addition to the fourmiRNAs described above,miR-638 was markedly down-regulated in CRC tissues.The expression levels of miR-638 were decreased in 83.33%the samples(30/36;Figure 1B,Additional file 3:Table S1b)and a 22.98%de-crease in expression in the CRC tissue samples comparedwith adjacent noncancerous tissue samples(2.323 to 1.789,p0.0001;Figure 1A).And a 27.28%decrease in moder-ately differentiated samples and 61.29%decrease in poorlydifferentiated samples compared to well-differentiatedsamples(Figure 1C).The miR-638 levels in all four CRCcell lines(HCT116,LoVo,SW1116,and SW480)weredownregulated compared with that of normal colorec-tal tissues(Figure 1D).These results demonstrate thatmiR-638 showed reduced expression levels in CRC andwas inversely correlated with tumor differentiation.miR-638 suppresses cell invasion and migrationTo understand the biological effect of miR-638 de-regulation on the development of colorectal carcin-oma,gain-or loss-of-function analyses were performedusing an overexpression or silencing strategy through thetransfection of miR-638 mimics or antagomiRs(usingan Amaxa Nucleofector device)into the CRC cell linesMa et al.Molecular Cancer 2014,13:118Page 3 of 13http:/www.molecular- and SW1116(the miR-638 levels in the CRCcells were confirmed through qRT-PCR;Figure 2Aand 2B).miR-638 elicited an apparent effect on the cellmotility of CRC cells.Matrigel-coated(for invasion)and-uncoated(for migration)transwell assays wereused to determine the invasiveness and migration ofHCT-116 and SW1116 cells after transfection withmiR-638 mimics or antagomiR-638.miR-638 overex-pression reduced the number of invasive HCT-116and SW1116 cells by 37.6%and 43.0%,respectively;however,antagomiR-638 enhanced cell invasion up to20.8%and 27.6%,respectively(Figure 2C,Additional file 4:Figure S1A).Moreover,miR-638 overexpression inhibitedHCT-116 and SW1116 cell migration by 23.4%and 33.1%,respectively,whereas the inhibition of miR-638 expressionenhanced cell migration up to 22.6%and 23.5%,re-spectively(Figure 2D,Additi