摘要目的 探讨微小RNA-137(miR-137)调控Notch1基因并介导自噬对肝癌细胞增殖和迁移的影响.方法 体外培养人SMMC7721肝癌细胞系,用miR-137模拟物、miR-137抑制物以及Notch1基因的干扰RNA(siRNA)转染细胞,分为正常对照组(NC组)、miR-137模拟物组、miR-137抑制物组、Notch1 siRNA组.采用实时定量-PCR (RT-PCR)检测SMMC7721细胞转染后miR-137与Notch1 mRNA的表达.细胞迁移实验(Transwell)分析miR-137及Notch1基因对SMMC7721细胞迁移侵袭的影响.细胞免疫组化观察SMMC7721细胞β-链蛋白(β-catenin)和波形蛋白(vimentin)的表达情况,自噬双标腺病毒检测SMMC7721细胞自噬体个数的变化.采用蛋白印迹法(Western blot)检测Notch1基因、上皮型钙黏蛋白(E-Cadherin)、神经型钙黏蛋白(N-Cadherin)、vimentin、选择性自噬接头蛋白(P62)及微管相关蛋白1轻链3(LC3)的表达.结果 实时荧光定量PCR (RT-PCR)结果显示miR-137抑制物组Notch1基因的相对表达含量(5.71±0.45)明显高于miR-137模拟物组(0.21±0.06),差异有统计学意义(P＜0.05).Transwell实验显示miR-137模拟物组(66.00 ±4.55)和Notch1 siRNA组(88.00 ±6.78)肝癌细胞侵袭转移的个数较miR-137抑制物组(515.00±35.12)明显降低(P＜0.05).细胞组化检测显示miR-137模拟物组及Notch1 siRNA组β-catenin表达明显增加且vimentin表达明显下降(P＜0.05).自噬双标腺病毒检测结果显示miR-137模拟物组自噬体数量(5.50 ±3.70)明显低于miR-137抑制物组(32.75±4.11),差异有统计学意义(P＜0.05).Western blot检测显示,与miR-137抑制物组、NC组比较,miR-137模拟物组Notch1基因、N-Cadherin、vimentin 和LC3表达水平明显降低,E-Cadherin、P62表达水平明显增高.Notch1 siRNA组Notch1基因、N-Cadherin、LC3表达水平明显低于NC组,E-Cadherin、P62表达水平明显高于NC组.结论 miR-137可通过抑制Notch1基因的表达抑制自噬从而抑制肝癌细胞的体外增殖、迁移和侵袭,有可能成为肝癌治疗的新靶点.

abstractsObjective To explore the role of miR-137 in the proliferation and migration of hepatocellular carcinoma (HCC) cells by regulating Notch1 and mediating autophagy.Methods The human SMMC7721 hepatoma cell line was transfected with miR-137 mimics,miR-137 inhibitor and Notch1 interfering RNA (siRNA),and divided into normal control group (NC group),miR-137 mimics group,miR-137 inhibitor group,Notch1 siRNA group.The expression levels of miR-137 and Notch1 mRNA after the transfection were detected by RT-PCR in SMMC7721 cells.Transwell experiments were performed to analyze the effect of miR-137 and Notch1 on the migration and invasion of SMMC7721 cells.The expression levels of β-catenin and vimentin in SMMC7721 cells were detected by immunohistochemistry.The number of autophagosomes was detected by double labeled adenovirus.Western blot was utilized to detect the expression of Notch1,E-Cadherin,N-Cadherin,vimentin,P62,and LC3.Results The results of RT-PCR showed that the relative expression level of Notch1 in miR-137 inhibitor group (5.71 ± 0.45) was significantly higher than that in miR-137 mimics group (0.21 ± 0.06) with statistical significance (P ＜ 0.05).The Transwell experiments showed that there were fewer invasive metastatic hepatoma cells in miR-137 mimics group (66.00 ± 4.55) and Notch1 siRNA group (88.00 ± 6.78) than that in the miR-137 inhibitor group (515.00 ±35.12) (P ＜0.05).The expression levels of β-catenin in miR-137 mimics group and Notch1 siRNA group were significantly increased and the expression level of vimentin was decreased (P ＜ 0.05).The results of autophagy double labeled adenovirus test showed that the number of autophagosomes in miR-137 mimics group (5.50 ± 3.70) was significantly fewer than that in miR-137 inhibitor group (32.75 ± 4.11),and the difference was statistically significant (P ＜ 0.05).The expression levels of Notch1,N-cadherin,vimentin,and LC3 protein in miR-137 mimics group were much lower than that in miR-137 inhibitor group and NC group,and the expression levels of E-Cadherin and P62 protein were greatly increased.The expression level of Notch1,N-cadherin,and LC3 protein in Notch1 siRNA group were significantly lower than that in NC group,and the expression levels of E-cadherin and P62 protein were much higher than that in NC group.Conclusion MiR-137 can inhibit the proliferation,migration and invasion of HCC cells by inhibiting the expression of Notch1 and autophagy,which may become a new target for the treatment of HCC.