The Regulatory Effects And The Mechanisms Of MiR-200b On Chemosensitivity Of Human Lung Adenocarcinoma Cells

Background and ObjectiveThe resistant phenotype of cancer cells results from both genetic and epigenetic dysregulation of key genes.Identified as important posttranscriptional regulators,microRNAs(miRNAs)cause either degradation or inhibition of gene translation through 3'-untranslated region(3'UTR)binding of target mRNAs,thereby involved in various biological and pathological processes such as proliferation,differentiation,morphogenesis,and carcinogenesis.The purpose of this study was to investigate the regulatory effecs of miRNA(s)on chemosensitivity of human lung adenocarcinoma cells via in vitro andin vivo models and to elucidate the underlying mechanisms through aspects such as cell proliferation,apoptosis,and cell cycle progress.Materials and Methods1.Cell morphologies of SPC-A1 and SPC-A1/DTX cells were observed under microscope.MTT assay was performed to detect the 50%inhibitory concentration(IC50)values for docetaxel.Colony formation assay was performed to detect the in vitro cell proliferating abilities.Flow cytometric analysis was performed to detect the apoptosis status and cell cycle distribution.2.MiRNA microarray analysis was performed to investigate the difference of miRNA profiles between SPC-A1 and SPC-A1/DTX cells.Real-time RT-PCR analysis was used to further confirm the result.3.Since miR-200b was the most down-regulated miRNA in SPC-A1/DTX cells in comparison with SPC-A1 cells,pri-miR-200b-gene-expressing plasmid(pPG/miR-200b),the negative control plasmid(pPG/NC),and miR-200b inhibitor were introduced into human lung adenoma SPC-A1,SPC-A1/DTX,and A549 cells through transient transfection.MTT assay was performed to detect the IC50 values for docetaxel.Colony formation assay was performed to detect the in vitro cell proliferating abilities.Flow cytometric analysis was performed to detect the apoptosis status and cell cycle distribution.4.pPG/miR-200b and pPG/NC were introduced into SPC-A1/DTX cells through transient transfection and subcutaneously into nude mice.Tumor growth was examined every other day.When the average tumor size reached about 50mm3,docetaxel was given through intraperitoneal injection.After 2 weeks,all mice were sacrificed.Transplanted tumors were excised and tumor tissues were used to perform hematoxylin and eosin(H&E)staining and proliferating cell nuclear antigen(PCNA)immunostaining analysis.5.Three different online miRNA databases(TargetScanHuman 6.0,DIANA-microT v3.0,and Microrna.org)were employed for prediction of miR-200b target genes.Dual luciferase activity assay was used to further confirm the result.6.Since transcription factor E2F3 was chosen as a preferred candidate target gene of miR-200b,E2F3 siRNA(siRNA/E2F3)and non-specific control siRNA(miRNA/NC)were introduced into SPC-A1/DTX cells through transient transfection.MTT assay was performed to detect the IC50 values for docetaxel.Colony formation assay was performed to detect the in vitro cell proliferating abilities.Flow cytometric analysis was performed to detect the apoptosis status and cell cycle distribution.7.A total of 53 lung adenocarcinoma tissues were collected from patients with advanced lung adenocarcinoma who have received docetaxel-based chemotherapies.Samples were divided into "sensitive"(complete response or partial response)and "insensitive"(stable disease or progressive disease)groups according to the patients' responses to the treatment.Real-time RT-PCR analysis was performed to detect the expression levels of miR-200b and E2F3 mRNA.Clinical information was co-considered to find the association between miR-200b expresion and prognosis.8.The online CpG predictive software MethPrime(http://www.urogene.org/methprimer/indexl.html)was employed for prediction of CpG islands nearby the transcription start site(TSS)of pri-miR-200b gene.9.SPC-Al/DTX cells were treated with different doses of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine(5-Aza-CdR),histone deacetylase inhibitor trichostatin A(TSA),or a combination of both.Real-time RT-PCR analysis was performed to detect changes of miR-200b expression level.10.Methylation-specific PCR(MSP)and gene sequencing were employed to detect the methylation status of pri-miR-200b TSS-related CpG islands in SPC-A1 and SPC-A1/DTX cells with or without 5-Aza-CdR treatment.Results1.Compared with parental SPC-A1 cells,SPC-A1/DTX cells are larger in size and have irregular distributions before cell fusion.SPC-A1/DTX cells were more resistant to docetaxel in comparison with SPC-A1 cells with the resistance index(RI)being 13.06.Colony formation assay revealed significant enhancement of proliferating ability of SPC-A1/DTX cells(p<0.01).Predominant accumulation in S phase was observed in SPC-A1/DTX cells compared with SPC-A1 cells(p<0.01)by flow cytometric analysis,while no significant deviation in apoptosis was observed(p>0.05).2.Based on miRNA microarray data,53 out of 470 miRNAs were found to be differentially expressed(more than 2-fold change)in SPC-A1/DTX cells compared to SPC-A1 cells,wherein miR-200b was the most down-regulated one(12.41-fold change).The result was validated by real-time quantitative RT-PCR(p<0.01).3.In both SPC-A1/DTX and A549 cells,ectopic miR-200b expression led to significant resensitization to docetaxel(p<0.01)3 suppression of the in vitro proliferating ability(p<0.01),a dramatic up-regulation of apoptosis rate(p<0.01),as well as an increased percentage of cells in G2/M phase(p<0.01)and a decreased population in S phase(p<0.01).In contrast,inhibited expression of miR-200b brought significant resistance to docetaxel to both SPC-A1 and A549 cells(p<0.01),and a mild enhancement of the in vitro proliferating ability to SPC-Al cells(p<0.05).4.Tumors derived from pPG/miR-200b-transfected SPC-A1/DTX cells grew substantially more slowly in comparison with the NC ones under the presure of docetaxel,indicating that ectopic miR-200b expression in SPC-A1/DTX cells could significantly enhance the in vivo response to docetaxel.Immunostaining analysis showed that the positive rate of proliferating cell nuclear antigen(PCNA)of tumors from pPG/miR-200b-transfected SPCA-1/DTX cells was decreased in comparison with that of the NC ones,indicating a down-regulation of the in vivo cell proliferating ability attributed to miR-200b.5.The transcription factor E2F3 was bioinformatically chosen as a preferred candidate target gene of miR-200b because of the two complementary sites of miR-200b in its 3'-untranslated region(3'-UTR)(position 2436-2442 and 2857-2863).The result was validated by dual luciferase activity assay.6.In SPC-A1/DTX cells,E2F3 knockdown notably induced resensitization to docetaxel(p<0.01),suppression of the in vitro proliferating ability(p<0.01),increased cell populations in both G1(p<0.01)and G2/M(p<0.05)phases and a decreased percentage of cells in S phase(p<0.01),but no enhancement of apoptosis(p>0.05).7.In clinical tissue samples,high expression level of miR-200b was found positively-correlated with both high chemosensitivity(Spearman rank test rho=0.77,p<0.01)and long overall survival(OS)(log-rank test,p<0.01)of the patients,while inversely-correlated with high expression of E2F3(Spearman rank test rho=-0.68,p<0.01).8.Pri-miR-200b-coding gene was located in chromosome:1091347-1093441,with its TSS region in chromosome:1092347-1092441(95 bp).Two CpG islands(CpG window size=200 bp,GC%>50%,observed CpG/expected CpG>0.6)were predicted within the region from 1000 bp upstream the TSS to 1000 bp downstream the TSS.9.SPC-A1/DTX cells showed significant re-expression of miR-200b after treatment of 5-Aza-CdR(p<0.01),TSA(p<0.01),and both(p<0.01),indicating the potential role of epigenetic mechanisms in the dysregulation of miR-200b in SPC-A1/DTX cells.10.The TSS-related CpG island of pri-miR-200b gene was fully methylated in both SPC-A1 and SPC-A1/DTX cells,and the methylation status was unchanged after treatment of 5-Aza-CdR,indicating that DNA methylation may not be the direct cause of the down-regulation of miR-200b in chemoresistant lung adenocarcinoma cells.Conclusions1.Our data indicate that miR-200b could function as a restorer of docetaxel chemosensitivity in human lung adenocarcinoma cells both in vitro and in vivo,causing cell proliferation suppression,apoptosis enhancement and G2/M arrest in cell cycle progress.2.The underlying mechanisms of miR-200b-mediated chemosensitization may be associated with the negative regulation of transcription factor E2F3,which is critical for the maintenance of normal cell cycle progression.3.DNA methylation and/or histone acetylation may be important epigenetic regulators controling miR-200b expression in chemoresistant lung adenocarcinoma cells.4.To our knowledge,this study is the first to show the dysregulation of miR-200b/E2F3 axis in human lung adenocarcinoma and its association with docetaxel sensitivity,indicting the posiblity of targeting miR-200b/E2F3 axis to reverse lung adenocarcinoma chemoresistance in clinic.