[Objectives]To explore the effects and mechanism of miR-15a-5p on oxaliplatin resistance in colorectal cancer HCT116/L cells.[Methods]The expression of miR-15a-5p in colorectal cancer sensitive cells HCT116 and resist...[Objectives]To explore the effects and mechanism of miR-15a-5p on oxaliplatin resistance in colorectal cancer HCT116/L cells.[Methods]The expression of miR-15a-5p in colorectal cancer sensitive cells HCT116 and resistant cells HCT116/L was detected by RT-qPCR method;the effect of oxaliplatin on the proliferation of HCT116 and HCT116/L cells was detected by MTT,and its IC_(50) and drug resistance fold of HCT116/L cells were calculated;the expressions of Wnt3a,β-Catenin and P-gp in HCT116 and HCT116/L cells were detected by Western Blot method.HCT-116/L cells were divided into 5 groups:blank control group HCT116/L,control group 1 transfected with miR-15a-5p mimics NC,experimental group 1 transfected with miR-15a-5p mimics,control group 2 transfected with miR-15a-5p inhibitor NC,and experimental group 2 transfected with miR-15a-5p inhibitor.The expression of miR-15a-5p in each group was detected by RT-qPCR method and the transfection efficiency was detected.The effect of different concentrations of oxaliplatin on the proliferation of cells in each group after transfection was detected by MTT and the half inhibitory concentration(50%inhibiting concentration,IC_(50))was calculated.The expressions of Wnt3a,β-catenin and P-gp in each group after transfection were detected by Western Blot method,and the expressions of Wnt3a,β-catenin and MDR1 mRNA in each group after transfection were detected by RT-qPCR method.[Results](i)RT-qPCR results showed that the expression of miR-15a-5p in HCT116/L cells was(0.16±0.05)significantly lower than that in HCT116 cells(P<0.05).(ii)The IC50 of oxaliplatin for HCT-116 cells and HCT116/L cells detected by MTT method were(13.51±2.62)and(103.08±12.29)μg/mL,respectively.The drug resistance index of HCT116/L was 7.63.(iii)Western Blot results showed that the expressions of Wnt3a,β-catenin and P-gp in HCT116/L cells were significantly higher than those in HCT-116 cells(P<0.05).(iv)After successful transfection,the IC50 of miR-15a-5p mimics group to oxaliplatin decreased to(40.78±2.47)μg/mL by MTT method,and its sensitivity to the drug was significantly improved compared with the control group.Western Blot results showed that the relative expressions of P-gp,Wnt3a andβ-catenin were significantly down-regulated(all P<0.05);RT-qPCR results showed that the relative expressions of MDR1,Wnt3a andβ-catenin mRNA were significantly down-regulated(all P<0.05).(v)After successful transfection,the expression of miR-15a-5p in the miR-15a-5p inhibitor transfection group was(0.38±0.04);MTT results showed that its IC50 for oxaliplatin was up-regulated to(132.77±7.97)μg/mL,and its sensitivity to chemotherapy drugs was significantly lower than that of the control group;Western Blot results showed that the relative expressions of P-gp,Wnt3a andβ-catenin were significantly up-regulated(all P<0.05);RT-qPCR results showed that the relative expressions of MDR1,Wnt3a andβ-catenin mRNA were significantly up-regulated(all P<0.05).[Conclusions]Up-regulation of miR-15a-5p expression can reverse the resistance of HCT116/L cell line to oxaliplatin.Up-regulation or down-regulation of miR-15a-5p will affect the expression of P-gp and Wnt/β-catenin signaling pathway-related proteins,suggesting that the mechanism of miR-15a-5p reversal of drug resistance may be related to the inhibition of Wnt/β-catenin pathway,thereby down-regulating the expression of P-gp.展开更多
文摘[Objectives]To explore the effects and mechanism of miR-15a-5p on oxaliplatin resistance in colorectal cancer HCT116/L cells.[Methods]The expression of miR-15a-5p in colorectal cancer sensitive cells HCT116 and resistant cells HCT116/L was detected by RT-qPCR method;the effect of oxaliplatin on the proliferation of HCT116 and HCT116/L cells was detected by MTT,and its IC_(50) and drug resistance fold of HCT116/L cells were calculated;the expressions of Wnt3a,β-Catenin and P-gp in HCT116 and HCT116/L cells were detected by Western Blot method.HCT-116/L cells were divided into 5 groups:blank control group HCT116/L,control group 1 transfected with miR-15a-5p mimics NC,experimental group 1 transfected with miR-15a-5p mimics,control group 2 transfected with miR-15a-5p inhibitor NC,and experimental group 2 transfected with miR-15a-5p inhibitor.The expression of miR-15a-5p in each group was detected by RT-qPCR method and the transfection efficiency was detected.The effect of different concentrations of oxaliplatin on the proliferation of cells in each group after transfection was detected by MTT and the half inhibitory concentration(50%inhibiting concentration,IC_(50))was calculated.The expressions of Wnt3a,β-catenin and P-gp in each group after transfection were detected by Western Blot method,and the expressions of Wnt3a,β-catenin and MDR1 mRNA in each group after transfection were detected by RT-qPCR method.[Results](i)RT-qPCR results showed that the expression of miR-15a-5p in HCT116/L cells was(0.16±0.05)significantly lower than that in HCT116 cells(P<0.05).(ii)The IC50 of oxaliplatin for HCT-116 cells and HCT116/L cells detected by MTT method were(13.51±2.62)and(103.08±12.29)μg/mL,respectively.The drug resistance index of HCT116/L was 7.63.(iii)Western Blot results showed that the expressions of Wnt3a,β-catenin and P-gp in HCT116/L cells were significantly higher than those in HCT-116 cells(P<0.05).(iv)After successful transfection,the IC50 of miR-15a-5p mimics group to oxaliplatin decreased to(40.78±2.47)μg/mL by MTT method,and its sensitivity to the drug was significantly improved compared with the control group.Western Blot results showed that the relative expressions of P-gp,Wnt3a andβ-catenin were significantly down-regulated(all P<0.05);RT-qPCR results showed that the relative expressions of MDR1,Wnt3a andβ-catenin mRNA were significantly down-regulated(all P<0.05).(v)After successful transfection,the expression of miR-15a-5p in the miR-15a-5p inhibitor transfection group was(0.38±0.04);MTT results showed that its IC50 for oxaliplatin was up-regulated to(132.77±7.97)μg/mL,and its sensitivity to chemotherapy drugs was significantly lower than that of the control group;Western Blot results showed that the relative expressions of P-gp,Wnt3a andβ-catenin were significantly up-regulated(all P<0.05);RT-qPCR results showed that the relative expressions of MDR1,Wnt3a andβ-catenin mRNA were significantly up-regulated(all P<0.05).[Conclusions]Up-regulation of miR-15a-5p expression can reverse the resistance of HCT116/L cell line to oxaliplatin.Up-regulation or down-regulation of miR-15a-5p will affect the expression of P-gp and Wnt/β-catenin signaling pathway-related proteins,suggesting that the mechanism of miR-15a-5p reversal of drug resistance may be related to the inhibition of Wnt/β-catenin pathway,thereby down-regulating the expression of P-gp.
