摘要
目的:探讨天然反义转录本Tetraspanin31调控CDK4的分子机制。方法:针对TSPAN31设计合成靶向干扰的siRNA,转染肝癌细胞,qRT-PCR和免疫印迹检测肝癌细胞实验组与对照组细胞CDK4的转录和翻译水平,免疫印迹检测细胞周期蛋白Cyclin D1、转录因子E2F1蛋白表达水平;在HEK293T细胞、Hela细胞中转染过表达TSPAN31全长、编码区及3’末端非翻译区质粒,qRT-PCR及免疫印迹检测CDK4基因转录和翻译水平。结果:与对照组相比,肝癌细胞siRNA干扰后TSPAN31蛋白相对表达量显著降低,CDK4相对表达量显著升高,Cyclin D1、E2F1蛋白相对表达量升高(均P<0.05)。与对照组相比,HEK293T细胞、Hela细胞转染过表达TSPAN313’末端非翻译区质粒组及过表达TSPAN31全长质粒组CDK4转录及翻译的相对表达水平显著降低(均P<0.05),而转染过表达TSPAN31编码区质粒组没有显著差异(均P>0.05)。结论:TSPAN31可在肝癌细胞中调控信号通路蛋白Cyclin D1、CDK4及E2F1蛋白表达,TSPAN31对CDK4的调控作用可能主要是通过转录产物的3’末端非翻译区进行的。
Objective:To investigate the molecular mechanism underlying natural antisense transcript Tetraspanin31(TSPAN31)regulation of CDK4.Methods:TSPAN31-targeted siRNA was designed,synthesized and transfected into hepatoma cells.qRT-PCR and Western blotting were used to detect the transcription and translation levels of CDK4 in experimental and control groups of hepatocellular carcinoma cells.The protein expression levels of cell cycle protein Cyclin D1 and transcription factor E2 F1 were detected by western blotting.The HEK293 T cells and Hela cells were transfected with plasmids harboring over-expressed full-length TSPAN31 or 3′-terminal non-translated region of TSPAN31.qRT-PCR and Western blotting were used to detect CDK4 gene transcription and translation levels.Results:Compared with the control group,the siRNA-interfered hepatocarcinoma cells showed significantly reduced relative expression level of TSPAN31 protein,higher relative expression levels of CDK4,Cyclin D1 and E2 F1 proteins(allP<0.05).Compared with control group,the relative expression levels of CDK4 transcription and translation were significantly reduced in HEK293 T and Hela cells transfected with the over-expressed 3′-terminal non-translated region of TSPAN31 or full-length TSPAN31(both P<0.05),but not in those transfected over-expressed coding region of TSPAN31(allP>0.05).Conclusion:TSPAN31 may regulate the expression of signal pathway proteins Cyclin D1,CDK4 and E2 F1 in hepatocellular carcinoma cells.TSPAN31 regulates CDK4 possibly via the 3’-terminal non-translated region of the transcription product.
作者
吕敏佳
古蕾
陈子鹏
王江林
孙雪萌
赵青
Lyu Minjia;Gu Lei;Chen Zipeng;Wang Jianglin;Sun Xuemeng;Zhao Qing(Department of Biochemistry and Molecular Biology,Guangzhou Medical University,Guangzhou,Guangdong 511436;Department of Clinical Medicine,Guangzhou Medical University,Guangzhou,Guangdong 511436,China)
出处
《广州医科大学学报》
2019年第6期6-11,共6页
Academic Journal of Guangzhou Medical University
基金
国家自然科学基金(81372466).
