miR-495通过抑制RNF113A的表达调控食管鳞癌细胞恶性表型

Regulation of malignant phenotype of esophageal squamous carcinoma cells by miR-495 via suppressing the expression of RNF113A

目的:探讨微小RNA-495(miR-495)对食管鳞癌细胞恶性表型的影响及分子机制。方法:在食管鳞癌细胞Eca109中转染miR-495模拟物、miR-495抑制剂及相应的随机对照序列,分别采用实时荧光定量PCR(qPCR)检测miR-495的表达,CCK-8试剂盒检测细胞增殖能力,流式细胞术检测细胞周期及凋亡率变化,Transwell实验检测细胞的侵袭和迁移情况,并用qPCR和Western blot检测下游靶基因RNF113A mRNA和蛋白的表达。结果:与对照组比较,转染miR-495模拟物组Eca109细胞中miR-495 mRNA水平显著升高(P<0.05),转染miR-495抑制剂组Eca109细胞中miR-495 mRNA水平显著降低(P<0.05);各组间细胞增殖能力无显著变化(P>0.05);与对照组比较,转染miR-495模拟物组细胞凋亡增多(P<0.01),迁移和侵袭能力下降(P<0.01),细胞被阻滞在G0/G1期(P<0.01);转染miR-495抑制剂组细胞凋亡减少(P<0.01),迁移和侵袭能力升高(P<0.01),进入细胞周期S期的细胞显著增多(P<0.05)。与对照组比较,转染miR-495模拟物组Eca109细胞中RNF113A蛋白水平表达下调(P<0.05),转染miR-495抑制剂组RNF113A蛋白水平表达上调(P<0.05),而RNF113A mRNA水平无显著变化(P>0.05)。结论:miR-495可促进Eca109细胞凋亡,抑制其迁移和侵袭能力,并调控细胞周期分布,miR-495可能通过调控RNF113A蛋白表达介导食管鳞癌细胞的恶性表型变化。

OBJECTIVE:To explore effects of microRNA-495(miR-495)on malignant phenotype of esophageal squamous carcinoma cells and its molecular mechanisms.METHODS:miR-495 mimics and miR-495 inhibitors,and the corresponding random sequence control were transfected into esophageal cancer cells Eca109.Real-time fluorescent quantitative polymerase chain reaction PCR(qPCR)was used to detect expression of miR-495,CCK-8 to detect cell proliferation,flow cytometry to detect cell cycle and apoptosis,transwell to detect cell migration and invasion ability,qPCR and western blot to detect expression of mRNAs and proteins of downstream target gene RNF113A.RESULTS:Compared with the control group,the miR-495 mRNA levels in the miR-495 mimics transfected group were significantly increased(P<0.05),and in the miR-495 inhibitor transfected group was significantly decreased(P<0.05).There was no significant changes in cell proliferation among the groups(P>0.05).The migration and invasion numbers in the miR-495 mimics transfected group were significantly decreased(P<0.01),and the apoptosis rate was significantly increased(P<0.01).The levels of cells blocked in the G0/G1 phase(P<0.01)were significantly more than that in the controls.The migration and invasion numbers in the miR-495 inhibitor transfected group were significantly increased(P<0.01),and the apoptosis rate was significantly decreased(P<0.01).The proportion of S phase cells in the miR-495 inhibitor transfected group was increased(P<0.05),and the difference was statistically significant.Compared with the control group,expression of the RNF113A protein in the miR-495 mimics transfected group was down-regulated(P<0.05),and expression of the RNF113A in the miR-495 inhibitor transfected group was up-regulated(P<0.05).However,the mRNA levels of RNF113A did not change significantly(P>0.05).CONCLUSION:miR-495 promoted cell apoptosis,inhibited cell migration and invasion ability,and regulated cell cycle distribution.Thus,miR-495 may mediate malignant phenotype of esophageal squamous carcinoma cells by regulating expression of the RNF113A protein.