microRNA-29c在雷公藤内酯醇抗克唑替尼耐药H3122肺癌细胞增殖的作用及机制研究

Effect of microRNA-29c on proliferation inhibition induced by triptolide in crizotinib resistant H3122 NSCLC cells and its mechanism

目的探讨microRNA-29c在雷公藤内酯醇(TP)抗克唑替尼耐药肺癌细胞增殖的作用及机制研究。方法将克唑替尼耐药H3122分为miRNA-29c inhibitor+TP组、阴性对照miRNA(miRNA-NC)+TP组、TP组和对照组。采用MTT检测细胞存活率,AnnexinV-FITC试剂盒检测细胞凋亡,Western blot法检测Bax、Bcl-2、cleaved-caspase3、DNMT3a表达,RT-qPCR检测DNA甲基转移酶3(DNMT3)mRNA、miRNA-29c表达,定量甲基化特异性PCR(QM‐SP)法检测视黄酸受体(RARb)、RAS域家族蛋白1(RASSF1)、死亡相关蛋白激酶1(DAPK1)和p16 mRNA表达和甲基化状态。结果与对照组比较,TP组中,TP浓度为10 nmol/L、25 nmol/L和50 nmol/L时,miRNA-29c表达增加(t分别=43.12、61.65、83.50,P均<0.05),DNMT3a表达下降(t分别=146.56、628.05、653.16,P均<0.05)。与对照组比较,TP组细胞存活率、p16、RASSF1、DAPK1、RARb甲基化降低(t分别=14.76、94.52、103.08、67.15、72.22,P均<0.05)、细胞凋亡率增加(t=-9.32,P<0.05),Western blot结果显示DNMT3a、Bcl-2表达明显降低,Bax、cleaved-ca‐spase3表达明显增高。与miRNA-NC+TP组比较,miRNA-29c inhibitor+TP组细胞存活率、p16、RASSF1、DAPK1、RARb甲基化明显增加(t分别=-4.08、-7.77、-67.84、-59.66、-46.79,P均<0.05),细胞凋亡率明显下降(t=5.40,P<0.05)。Western blot结果显示DNMT3a、Bcl-2表达明显增加,Bax、cleaved-caspase3表达明显降低。结论 TP能够增加miRNA-29c表达,下调DNMT3表达和活性,降低抑癌基因的甲基化水平,抑制H3122CR细胞增殖,发挥抗肺癌作用。

Objective To investigate the role of microRNA-29 c in proliferation inhibition induced by triptolide(TP in crizotinib resistant H3122 non-small cell lung cancer(NSCLC)cells and its mechanism. Methods The H3122 cells were divided into four groups:miRNA-29 c inhibitor +TP group,miRNA negative control(NC)+TP group,TP group and control group.The cell survival rate was detected by MTT.The apoptosis was detected by AnnexinV/PI.Western blot was used to detect the expression of Bax,Bcl-2,cleaved-caspase3 and DNMT3 a.The expression of DNMT3 a mRNA and microRNA-29 c were detected by RT-qPCR.The methylation of RARb,RASSF1,DAPK1 and p16 were detected by quantitative methylation-specific PCR(QMSP). Results Compared with control group,the expression of microRNA-29 c increased in TP group when the concentration of TP was 10 nmol/L,25 nmol/L and 50 nmol/L(t =43.12,61.65,83.50,P<0.05),while the DNMT3 a expression decreased(t=146.56,628.05,653.16,P<0.05).Compared with control group,the cell survival rate,methylation of p16,RASSF1,DAPK1,RARb decreased(t=14.76,94.52,103.08,67.15,72.22,P<0.05),while the apoptosis rate increased in TP group(t=-9.32,P<0.05). Western blot result showed that DNMT3 a and Bcl-2 expressions decreased,Bax and cleaved-caspase3 expressions increased in TP group.Compared with miRNA-NC+TP group,the cell survival rate,methylation of p16,RASSF1,DAPK1,and RARb increased in miRNA-29 c inhibitor+ TP group(t =-4.08,-7.77,-67.84,-59.66,-46.79,P<0.05),but the apoptosis rate decreased(t=5.40,P<0.05).Western blot result showed that DNMT3 a,Bcl-2 expression increased,Bax and cleaved-caspase3 expression decreased in the microRNA-29 c inhibitor +TP group. Conclusion TP inhibits the proliferation of H3122 CR cells via up-regulating the expression of microRNA-29 c and down-regulating the expression and activity of DNMT3,resulting in tumor suppressor gene methylation.