Screening And Validation Of MiRNA Expression In TGF-? Induced EMTed H358 Cells

Objective:Non-small cell lung cancer?NSCLC?is the leading cause of cancer death.Epithelial-to-mesenchymal transition?EMT?plays an important biological role in cancer progression,metastasis,radiation and chemotherapy resistance.Thus,exploring the mechanism of EMT is clinically important for the diagnosis and therapy for the malignancy.MicroRNAs?miRNAs?are a class of small noncoding RNAs that have remarkably stable form,which are emerging as specific biomarkers for early diagnosis,prognosis,and drug resistance of cancers.Our previous data indicated that H358 cells underwent EMT by continuously treated with TGF-?1.In this study,we used miRNA sequencing to comprehensively characterize miRNA profiles in H358 and H358-TGF-?cells.Through establishing miRNAs-target gene regulatory networks,the possible role of miRNAs in NSCLC cell EMT was analyzed.Methods:1.Cell culture and treatmentHuman Non-small cell lung cancer?NSCLC?cell line H358,and H358-TGF-?cells obtained through 5ng/mL TGF-?1 treatment,were maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum,100U/mL penicillin and 100U/mL streptomycin.Both of the cells were incubated in a humidified atmosphere of 5%CO₂ in air at 37?C.2.Cell transfectionMiRNA mimics and NC miRNAs were transfected into H358 and H358-TGF-?cells,and the cells were divided into the negative control group?NC?and the miRNA mimics transfection groups.3.Real-time PCR assayTotal RNA was extracted from the above cells.The expressions of E-cadherin and GLI3 mRNA in each group were detected by Real-time PCR.The miRNAs expression was detected according to the instruction of RT-PCR assay from Ruibo.4.MiRNAs sequencingThis method is provided by kangcheng Company of Shanghai.Total RNA of each sample was used to prepare the miRNA sequencing library.The libraries were denatured as single-stranded DNA molecules,captured on Illumina flow cells,amplified in situ as clusters and finally sequenced for 50cycles on Illumina NextSeq per the manufacturer's instructions.After sequencing,the Solexa CHASTITY quanlity filtered reads were harvested as Clean Reads.MiRNA target prediction was performed by targetscan and other popular method,then the GO and KEGG pathway analysis was performed based on the top 10 differentially expressed miRNAs.5.GEO data analysisTwo microarray data of H358 cells treated by TGF-??GSE49644 and GSE79235?,were downloaded from GEO datasets to analyze the expression of GLI3 mRNA in the EMT.6.Western blotTotal protein was extracted from cells in each group and the expressions of E-cadherin or GLI3 protein were detected by Western blot assay.7.Statistical analysisSPSS Statistics 17.0 software was used to analyze the statistical significance.Data are presented as mean±SD.Statistical comparisons between experimental groups were analyzed by?2 and two-sample t-test.P<0.05 was taken to indicate statistical significance.Results:1 Differentially expressed miRNAs in H358 and H358-TGF-?cells by miRNA sequencing1.1 Results of miRNA sequencingConmpared to H358 cells,88 miRNAs were up-regulated and 71miRNAs were significantly down-regulated in H358-TGF-?cells.The FC values of the top ten up-regulated miRNAs was 14.37²37.51,while the down-regulated ratio of the first ten miRNAs was 84%⁹8%.1.2 qPCR validation the results of miRNA sequencingFour significant miRNAs were selected for PCR validation.Compared with H358 cells,the expression of miR-381-3p and miR-146a-5p were up-regulated,while the expressions of miR-200c-3p and miR-203a-3p were significantly inhibited,which were consistent with the sequencing results.2 Prediction and validation of target genes by Top 10 up-regulated miRNAs2.1 Prediction of the possible target genes by Top 10 up-regulated miRNAAlthough each miRNA can target dozens to hundreds of target genes,few target genes that are co-regulated by the above miRNAs.It as found that miR-146a-5p,miR-411-5p,miR-495-3p,and miR-654-3p all have possible binding sites in epithelial marker CDH1,suggesting that these miRNAs might involve in EMT through regulation of CDH1 expression.2.2 Effect of miRNAs on the expression of E-cadherin in H358 cellsMiR-146a-5p,miR-411-5p,miR-495-3p,and miR-654-3p mimics were transfected into H358cells.Western blot assay indicated that miR-411-5p could inhibit the expression of E-cadherin at protein level.3 Prediction and validation of target genes by Top 10 down-regulated miRNAs3.1 Prediction of the possible target genes by Top 10 down-regulated miRNAThree prediction software all indicated that miR-203-3p,miR-200c-3p and miR-7-5p might regulate one common target gene-GLI3,suggested that the three significantly down-regulated miRNAs in EMTed H358-TGF-?cells might regulate GLI3 expression through bind to the 3'-UTR region.3.2 Validation the miRNAs expression in H358-TGF-?cellsCompared with H358 cells,the expression levels of miR-200 family-miR-200c-3p/200b-3p/429,were significantly decreased in H358-TGF-?EMT cells.In addition,the expression of miR-203a-3p was significantly decreased.The results were consistent with the sequencing results.3.3 GLI3 expression in EMT cellsThe expression of GLI3 protein in H358-TGF-?cells was significantly higher than that in H358 cells,suggesting that GLI3 may be a mesenchymal marker.3.4 Effect of miRNAs on GLI3 expression in H358-TGF-?cellsThe expression of GLI3 was detected after miR-200c-3p/200b-3p/429,miR-203a-3p,and miR-7-5p mimics transfection in H358-TGF-?cells.PCR results showed that the above five miRNAs could significantly inhibit GLI3mRNA expression.Western blot assay further indicated that these miRNAs could inhibit the expression of GLI3 at protein level when transfected alone or in combination.Conclusion:1.MiRNAs expression profile is significantly changed in EMTed H358cells after long-term TGF-?1 treatment.88 miRNAs are upregulated and 71are downregulated in EMTed H358 cells,suggested that miRNAs are important molecular involved in the process of EMT.2.The top10 upregulated miRNAs related to EMT of H358-TGF-?cells are miR-381-3p,miR-146a-5p,miR-379-5p,miR-455-5p,miR-455-3,miR-411-5p,miR-654-3p,miR-495-3p,and miR-323a-3p,respectively.Among these miRNAs,miR-411-5p was shown to inhibit the expression of E-cadherin protein.3.The most significantly downregulated miRNAs associated with EMT in H358-TGF-?cells are miR-203-3p,miR-205-5p,miR-141-3p,miR-200c-3p,miR-34a-5p,miR-141-5,miR-95-3p,and miR-7-5p.Among them,miR-203-3p,200c-3p,and miR-7-5p alone or in combination,could inhibit GLI3 expression both at mRNA and ptotein level,suggested that miRNA-Hh/GLI3 signaling pathway was involved in regulation of EMT in NSCLC.