CRNDE介导miR-33a-5p/CDK6轴影响细胞周期调控人体肝癌细胞增殖的实验研究  

Experimental study on CRNDE mediated miR-33a-5p/CDK6 axis affecting cell cycle regulation and proliferation of human liver cancer cells

作　　者：梅小平 姚明 周清河 许浏 周鸿鲲 李彦 Mei Xiaoping

机构地区：[1]浙江省嘉兴市第一医院(嘉兴学院附属医院),314000

出　　处：《浙江临床医学》2023年第8期1117-1120,共4页Zhejiang Clinical Medical Journal

基　　金：浙江省嘉兴市科技计划项目(2022AD30068);浙江省中医药科技计划项目(2023ZL699)。

摘　　要：目的探讨CRNDE介导miR-33a-5p/CDK6轴影响细胞周期,调控人体肝癌细胞增殖的作用机制。方法运用细胞转染技术构建稳定干扰CRNDE/低表达CRNDE的人体肝癌细胞模型shRNA-CRNDE-HuH-7;RT-qPCR及Western blotting技术检测HL-7702人体肝细胞株、HuH-7人体肝癌细胞株及shRNA-CRNDE-HuH-7细胞株中CRNDE、miR-33a-5p及CDK6的表达情况,评估其相关性及miR-33a-5p/CDK6轴随CRNDE表达量变化的相关程度;对上述三组细胞株进行细胞增殖实验(CCK8)、细胞凋亡检测、细胞侵袭测试及细胞周期检测,评估在不同CRNDE表达情况下三组细胞株的增殖、凋亡、侵袭能力及细胞周期的变化。结果RT-qPCR及Western blotting技术检测shRNA-CRNDE-HuH-7细胞株中CRNDE表达低于HuH-7人体肝癌细胞株(P<0.05);当控制位点为CDK6时,三组细胞株中CRNDE表达量与其呈正相关(r=0.856,P<0.05);当控制位点为miR-33a-5p时,三组细胞株中miR-33a-5p表达量与CDK6及CRNDE均呈负相关(r=-0.883,P<0.05;r=-0.937,P<0.05);在三组细胞株中,随CRNDE表达下调,细胞增殖、侵袭能力及细胞周期均受到抑制(P<0.05),shRNA-CRNDE-HuH-7细胞株中肿瘤细胞凋亡高于HuH-7细胞株(P<0.05)。结论CRNDE与miR-33a-5p存在靶向关系,CRNDE以靶向调节作用抑制miR-33a-5p的表达,进而介导CDK6的升高,通过miR-33a-5p/CDK6轴促进肝细胞肝癌的增殖,其可能成为肝细胞肝癌的新肿瘤标志物和分子生物治疗靶点。Objective To explore the mechanism of CRNDE mediated miR-33a-5p/CDK6 axis affecting cell cycle and regulating the proliferation of human liver cancer cells.Methods The cell transfection technology was used to construct a stable human liver cancer cell model shRNA CRNDE HuH-7 that interferes with CRNDE/low expression of CRNDE.RT qPCR and Western blotting techniques were used to detect the expression of CRNDE,miR-33a-5p,and CDK6 in HL-7702 human liver cell line,HuH-7 human liver cancer cell line,and shRNA CRNDE HuH-7 cell line,and to evaluate their correlation and the degree to which the miR-33a-5p/CDK6 axis changes with CRNDE expression levels.The cell proliferation experiments(CCK8),apoptosis detection,invasion testing,and cell cycle testing on the three groups of cell lines in the appeal were conducted,and the changes in proliferation,apoptosis,invasion ability,and cell cycle of the three groups of cell lines under different CRNDE expression conditions was evaluated.Results The expression of CRNDE in shRNA CRNDE HuH-7 cell line was lower than that in HuH-7 human liver cancer cell line using RT-qPCR and Western blotting techniques,with statistical significance(P<0.05).When the control site was CDK6,the expression level of CRNDE in the three cell lines was positively correlated with it(r=0.856,P<0.05).When the control site was miR-33a-5p,the expression of miR-33a-5p was negatively correlated with CDK6 and CRNDE in all three cell lines(r=-0.883,P<0.05;r=-0.937,P<0.05).In the three cell lines,with the downregulation of CRNDE expression,cell proliferation,invasion ability,and cell cycle were inhibited(P<0.05),and tumor cell apoptosis was higher in the shRNA CRNDE HuH-7 cell line than in the HuH-7 cell line,with statistical significance(P<0.05).Conclusion There is a targeting relationship between CRNDE and miR-33a-5p.CRNDE sponges on miR-33a-5p in the form of ceRNA,which in turn mediates the increase of CDK6.It promotes the proliferation of hepatocellular carcinoma through the miR-33a-5p/CDK6 axis,and may become a new

关 键 词：CRNDE miR-33a-5p 肝细胞肝癌 细胞周期蛋白依赖性蛋白激酶6 

分 类 号：R735.7[医药卫生—肿瘤]