Screening And Functional Study Of Differentially Expressed LncRNAs In Carboplatin-Resistant Ovarian Cancer Cells

Research background : Ovarian cancer is one of the three common malignant tumors in the female reproductive system.It has the characteristics of high mortality and morbidity and low survival rate,which seriously threatens women’s life and health.Due to the insidiousness of the early-onset in ovarian cancer,it is usually diagnosed in the terminal stage.The major treatment approach was mainly rely on surgical removal of lesion and postoperative chemotherapy in ovarian caner currently.Carboplatin one of the commonly chemotherapeutic drug in treating ovarian cancer has easily exerted an influence on resistance in ovarian cancer with the prolongation of chemotherapy,and producing poor prognosis.Therefore,studying the resistant-regulated mechanism and funding out the critical factors is the essential way to cure ovarian cancer clinically.Research objectives:Long non-codingRNAs are a class of non-codingRNAs whose length is greater than 200 bp.Previous studies have shown that the differential expression of lncRNAs is related to the growth of a variety of carcinoma and drug resistance,but the involvement of lncRNAs in the regulation of carboplatin resistance in ovarian cancer has rarely been reported.Based on the study of expression profiling chip,our research,mainly wanted to find out the molecular mechanism of lncRNAs regulating carboplatin resistance in ovarian cancer comprehensively,and identifies key lncRNAs and performs related functional verification.Preliminarily elucidate and further complete the molecular mechanism of lncRNA regulating carboplatin resistance in ovarian cancer.Research methods:1.Expression profile chip and bioinformatics was used to analyze the lncRNA profile of 2 pairs of carboplatin-resistant and sensitive cells: using SBC Human(4*180K)lncRNA microarray to screen ovarian cancer sensitive cells Hey A8,SKOV3 and drug-resistant cells Hey A8-CBP,SKOV3-CBP differentially express lncRNA and mRNA.From the aspects of gene ontology annotation,disease type classification,KEGG pathway enrichment and targeted mRNA,the molecular mechanism of lncRNA regulating drug resistance in ovarian cancer is analyzed in general.2.Screening of common differentially expressed lncRNAs in 2 pairs of carboplatin-resistant ovarian cancer cells: through RT-q PCR technology,the above-screened ovarian cancer resistance-related lncRNA and target mRNA were been further verification in the 2 pairs of carboplatin-resistant ovarian cancer cells.One or two lncRNAs stably and differentially expressed in carboplatin-resistant ovarian cancer cells were screened out for further research.Eventually,lncRNA DAPK1-IT1 is determined as the subsequent research object.3.Research on the molecular mechanism of lncRNA DAPK1-IT1 regulating carbopaltin resistance in ovarian cancer: constructed lncRNA DAPK1-IT1 overexpression ovarian cancer cell line via lentiviral transfection,and the overexpression of lncRNA DAPK1-IT1 was verified by in vitro cell function experiments The phenotypic changes in carboplatin-resistant ovarian cancer cells preliminarily revealed that lncRNA DAPK1-IT1 is involved in the molecular mechanism of regulating drug resistance in ovarian cancer.Results : 1.Based on the expression profile chip and bioinformatics analysis of carboplatin resistance in ovarian cancer cells express,a total of a of 3239 differential lncRNAs were analyzed,via GO enrichment analysis,KEGG pathway enrichment analysis and Venn diagram analysis.Obtained 161 lncRNAs differentially expressed in 2 pairs of ovarian cancer carboplatin-resistant cells,including 107 lncRNAs up-regulated in 2 pairs of ovarian cancer carboplatin-resistant cells,and 54 lncRNAs down-regulated.2.RT-q PCR verified the expression of co-express differentially expressed lncRNA in two pairs of carboplatin-resistant ovarian cancer cells.Combining with the expression profile analysis results and RT-q PCR results,we finally selected lncRNA DAPK1-IT1 as the subsequent study lncRNA.3.Research on the mechanism of lncRNA DAPK1-IT1 in regulating carboplatin resistance in ovarian cancer.The results of clone formation experiment without treating with carboplatin showed that overexpression of lncRNA DAPK1-IT1 inhibited the growth of Hey A8-CBP,but had no effect on the growth of SKOV3-CBP.The cell cycle results showed that the overexpression of lncRNA DAPK1-IT1 in Hey A8-CBP can significantly block the cell cycle.And the results of drug-added clone formation showed that after overexpressing lncRNA DAPK1-IT1,the sensitivity of carboplatin was significantly increased in Hey A8-CBP and SKOV3-CBP.The cell cycle results of drug-added showed that after treatment with a certain concentration of carboplatin,the overexpression of lncRNA DAPK1-IT1 had little effect on the cell cycle of Hey A8-CBP cells,while the cell cycle of SKOV3-CBP was blocked in G2/M phase.And the Western blot results showed that after treating with carboplatin,the cyclin and autophagy proteins of the SKOV3-CBP ovarian cancer cells after overexpression of lncRNA DAPK1-IT1 changed to varying degrees.Conclusion:lncRNA DAPK1-IT1 was selected as the object of subsequent study by combining the analysis of the expression profile microarray of 2ovarian cancer carboplatin resistant cells and RT-q PCR expression verification.By constructing the overexpressed lncRNA DAPK1-IT1 ovarian cancer cell line with carboplatin resistance,we found that lncRNA DAPK1-IT1 can affect the proliferation of carboplatin-resistant ovarian cancer cells by blocking the G0/G1 phase of the cell cycle and inhibiting clone formation,and under the conditions of carboplatin treatment,it can inhibit the growth,and affect the expression of cell cycle-related and autophagy-related proteins in carboplatin-resistant ovarian cancer cells,thus thereby exerting anti-tumor effects and participating in the regulation of carboplatin resistance in ovarian cancer.Moreover,through bio-informatics analysis,lncRNA DAPK1-IT1 was enriched in multiple signaling pathways,suggesting that lncRNA DAPK1-IT1 is expected to be used as a potential target for the treatment of drug-resistant ovarian cancer,which has certain theoretical and practical value.