| Background and objective:Ovarian cancer is the first leading cause of mortality in gynecological malignancies,and the number of deaths due to ovarian cancer ranks fifth in cancer deaths among women,which is a serious threat to women’s lives and health.Early symptoms of ovarian cancer is not obvious.The tumor has developed to the advanced stage before diagnosis.At present,the combination of surgery and chemotherapy is the common treatment for ovarian cancer.However,the 5-year survival rate of patients with advanced ovarian cancer is less than 30%,because more than 70%of patients become resistant to chemotherapy.miRNA is a kind of non-coding small RNA,which inhibits the translation of target genes by binding to the mRNA of target gene.As an important member of miRNA family,let-7i has been found to be involved in the development,metastasis and drug resistance of various types of tumors.This study was to analyze the expression level of let-7i in ovarian cancer and to explore the effect of let-7i on proliferation,migration and invasion as well as sensitivity to cisplatin of ovarian cancer cells.Besides,the target genes of let-7i were predicted and validated to investigate the mechanism of let-7i,the data mining was applied to identify the key genes in the development of ovarian cancer.Methods:1.Four cells were cultured:IOSE、SKOV3、SKOV3/DDP and A2780,and the A2780/DDP cells were induced by increasing progressively concentration and shock of high-dose cisplatin.Real-time PCR was applied to test the expression of let-7i in these cells.2.let-7i mimics、let-7i mimics control、let-7i inhibitor and let-7i inhibitor control were transfected into ovarian cancer cells by method of lipofection,and the efficiency of transfection was detected by real-time PCR.Cell proliferation,migration and invasion were measured by CCK-8 and EdU experiments,Transwell migration assay,Transwell invasion assay,respectively.The effect of let-7i on cell sensitivity to cisplatin was detected by MTT assay.3.TargetScan、miRanda and PicTar were applied to predict the target genes of let-7i,and the intersection of genes predicted by the three softwares were regarded as candidate targets.The analysis of gene ontology and pathway associated with these targets were conducted by DAVID database to identify potential pathway and key genes.4.The double fluorescent reporter gene experiment and western blot experiment were applied to verify candidate target genes and detect the effect of let-7i on enriched pathway.Besides,the OvMark database was applied to analyze the correlation of the expression level of target gene of let-7i with survival rate of ovarian cancer patients.5.Differentially expressed genes(DEGs)were identified from GSE36668、GSE18520.GSE14407 by GE02R tool.And the PPI network was constructed by STRING.Additionally,differentially expressed miRNAs were identified from GSE47841 by GE02R and the targets of miRNAs were predicted by miRecords.Kaplan-Meier plotter was conducted to analyze the correlation of the expression level of genes with survival rate of ovarian cancer patients.Results:1.A2780/DDP cell was constructed.And the expression level of let-7i in SKOV3 cells and A2780 cells were lower than that in IOSE cells(P<0.01),but was higher than that in SKOV3/DDP and A2780/DDP cells(P<0.01).2.The expression of let-7i was higher after transfection with let-7i mimics(P<0.01).The abilities of proliferation,migration and invasion of cells transfected with let-7i mimics were decreased compared to ones transfected with let-7i control(all P<0.05),and the IC50 of SKOV3/DDP cells decreased from 19.06μmol/L to 15.52μmol/L after transfection with let-7i mimics,while the IC50 of A2780/DDP cells decreased from 13.30μmol/L to 8.33μmol/L.3.A total of 324 genes were identified as the targets of let-7i by TargetScan、miRanda and PicTar.The result of bioinformatics data analysis indicated that the predicted target genes of let-7i were mainly enriched in MAPK pathway(P<0.05),in which TAB2 was involved.4.Double fluorescent reporter gene experiment indicated that TAB2 is the target gene of let-7i,and the results of western blot indicated that the protein level of TAB2 and TLR4 is decreased in SKOV3 and A2780 cells after transfection with let-7i mimics(P<0.05)and let-7i could decrease the level of P38 phosphorylation in SKOV3 cells and A2780 cells(P<0.05).Besides,the analysis by OvMark database revealed that overall survival and disease free survival of ovarian cancer patients with high mRNA level of TAB2 were shorter(P<0.05).5.A total of 345 DEGs were identified.The PPI network of DEGs consisted of 141 nodes,including 16 hub genes.Overall survival for patients with ovarian cancer was obtained according to the low and high expression of each gene.It was found that high mRNA expression of CCNB1(HR 1.26[1.01-1.58],P = 0.04)was associated with worse overall survival for ovarian cancer patients,as well as CENPF(HR 1.18[1.03-1.35],P = 0.014),KIF11(HR 1.15[1-1.31],P = 0.04),ZWINT(HR 1.15[1-1.31],P = 0.04).Conclusions:The expression of let-7i in ovarian cancer is decreased than normal ovarian cells,while it is higher than that in ovarian cancer cells resistant to cisplatin.Overexpression of let-7i can inhibit the growth,migration and invasion of ovarian cancer cells SKOV3 and A2780,can reverse the drug resistance to cisplatin of SKOV3/DDP and A2780/DDP cells.let-7i can inhibit the phosphorylation of P38,at least in part,by down-regulating TAB2 and TLR4,which may be the correspondent mechanism.In addition,we found that the expression of CCNB1,CENPF,KIF11,ZWENT were up-regulated in ovarian cancer,and the four genes could be used to predict the prognosis of ovarian cancer. |
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