Metformin-sensitized Cisplatin-resistant Ovarian Cancer-related MicroRNA Identification And Its Regulatory Signaling Pathway

Part ? Effects of Metformin and Cisplatin on the Proliferation of Ovarian Cancer Cell Lines under Different Glucose Concentration Culture ConditionsObjective: To explore the effect of different glucose concentration culture conditions on metformin combined with cisplatin to kill ovarian cancer cellsMethods: Real-time label-free cell analysis technology(RTCA)was used to detect the inhibitory effects of MET single drug,DDP single drug,and MET in combination with DDP on ovarian cancer cell lines SKOV3,A2780,SKOV3-GFP,SKOV3-GFP/DDP.Inhibition of ovarian cancer cell lines,using compusyn software to determine the correlation between the effects of MET and DDP through the size of the combination index.Results: Low glucose culture medium increased the inhibition of metformin on the proliferation of ovarian cancer cells SKOV3,A2780,SKOV3-GFP,SKOV3-GFP/DDP(P<0.001),and also enhanced the combination of metformin and cisplatin to kill SKOV3,A2780,SKOV3-GFP/DDDP(P<0.05),according to the use of compusyn software to determine the size of CI,under low glucose culture conditions,metformin and cisplatin can better play a synergistic effect.High glucose culture conditions may cause metformin to antagonize cisplatin.Conclusion: Low glucose culture conditions can help metformin improve the cisplatin's effect on killing ovarian cancer cells,while high glucose culture inhibits metformin's synergistic effect on cisplatin.Part ? Metformin-Sensitized Cisplatin-Related MiRNA and Identification of Its Downstream Regulatory PathwaysObjective: To identify miRNAs associated with MET-sensitized DDP inhibiting ovarian cancer cells,and to detect possible involvement of metformin-sensitized cisplatin-regulated pathways.Methods: Real-time fluorescence quantitative polymerase amplification(QRT-PCR)was used to compare the effects of single drug MET,DDP and MET in combination with DDP on the expression level of related miRNAs under different glucose concentrations.Bioinformatics analysis of related miRNAs may be involved in the regulation of proteins Pathway,predicting MET-sensitized DDP-related pathways,Western Blot(WB)verified the changes in the node proteins of this pathway.Result: QRT-PCR results showed that under low glucose concentration culture conditions,MET and DDP promoted to down-regulate the relative expression of miR-33 b,miR-214,miR-503 and miR-214 in ovarian cancer cell lines SKOV3,A2780,SKOV3-GFP/DDP Amount(P<0.05),and in SKOV3-GFP cells,the relative expression of miRNA increased.The results of bioinformatics analysis indicate that MET may suppress the platinum-based resistance of ovarian cancer cells by inhibiting miR-214-mediated TGF?-Wnt-epithelial-mesenchymal transition(EMT)circulating feedback network.WB results show that the relative In single drug MET and DDP,MET combined with DDP treatment can activate the AMPK pathway and inhibit m TOR,TGF-beta,WNT/?-Catenin protein pathway.Conclusion: Conclusion: The effect of MET-sensitized DDP on killing ovarian cancer cell lines may be achieved by down-regulating the relative expression levels of miR-33 b,miR-214,miR-503 and miR-129,while the relative expression level of miRNA in low glucose culture conditions decreased even more Obviously;MET participates in the regulation of protein pathways that miR-214 may target;MET can activate AMPK and inhibit TGF?/Wnt/m TOR signaling pathway to reverse cisplatin resistance.Part ? Exploring the Effects of MiR-214 Mimics & Inhibitors on the Anti-Ovarian Cancer Characteristics and Downstream Regulatory Pathways of Metformin-Sensitized CisplatinObjective: To investigate the effect of transient transfection of miR-214 mimics and inhibitors on MET-sensitized DDP anti-ovarian cancer-related regulatory pathways.Methods: The cell line SKOV3 was transiently transfected with miR-214 mimics and inhibitors.After transient transfection,the total RNA of the cells was extracted.After reverse transcription,QRT-PCR was used to verify the expression of miR-214 after transfection.Western Blot was used to compare miR-214 transfection.The effect of MET-sensitized DDP on Wnt/?-catenin signaling pathway before and after-214 mimics or inhibitors.Results: The results of QRT-PCR proved that the miR-214 inhibitor reduced the relative expression of miR-214,and the miR-214 mimetics increased the relative expression of miR-214,demonstrating that the transient was successful.Compared with the empty and blank groups,miR-214Wnt/?-catenin signaling pathway Wnt1,P-?-catenin and ?-catenin protein expression in the 214 mimetics group after MET compatible with DDP,transfection of miR-214 inhibitor can further inhibit the Wnt/?-catenin pathway inhibited by MET.Conclusion: MET reverses the cisplatin resistance of ovarian cancer caused by the Wnt/?-catenin pathway targeted by miR-214.