体外siRNA-GAS5干扰肝癌HepG2细胞生物学行为改变  

siRNA-GAS5 interferes HepG2 cells and changes in biological behavior in vitro

作　　者：黄海锋 葛勇胜 刘文斌 马金良 Huang Haifeng;Ge Yongsheng;Liu Wenbin(Dept of General Surgery,The Affiliated Provincial Hospital of Anhui Medical University,Hefei 230001)

机构地区：[1]安徽医科大学附属省立医院普外科,合肥230001

出　　处：《安徽医科大学学报》2018年第8期1210-1216,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81272398);安徽省公益性技术应用研究联动计划项目(编号:1604f0804011)

摘　　要：目的探究生长特异性调节转录因子5(GAS5)在肝癌组织表达及GAS5小干扰RNA(siRNA-GAS5)转染对HepG2肝癌细胞的侵袭增殖的影响,并进一步分析其可能的调控机制。方法选取手术肝癌切除的癌组织及其癌旁组织20例,qRT-PCR检测肝癌及其癌旁组织长链非编码RNA GAS5(GAS5)表达以及Western blot检测周期依赖性蛋白激酶6(CDK6)表达。利用脂质体转染siRNA-GAS5干扰HepG2细胞株中GAS5表达水平,划痕实验、Transwell和CCK-8检测转染后HepG2细胞的迁移、侵袭及增殖能力,流式细胞仪检测干扰后HepG2细胞凋亡周期变化。Western blot、qRT-PCR验证细胞周期调控蛋白周期素D1(Cyclin D1)、CDK6、E2F-1表达。结果肝癌组织中GAS5表达水平低于对应癌旁组织,CDK6表达高于癌旁组织。转染siRNAGAS5的HepG2细胞迁移侵袭增殖能力明显增强。转染siRNA-GAS5的HepG2细胞Cyclin D1、CDK6、E2F-1蛋白表达下降,干扰后的HepG2细胞周期发生G1/S期改变。结论GAS5在肝癌组织的表达出现下调;GAS5可明显抑制肝癌细胞迁移、侵袭及增殖能力;GAS5调控肝癌细胞周期变化可能是通过Cyclin D1/CDK6/E2F-1通路。Objective To study the expression and invasion of long non-coding RNA GAS5 on hepatocellular tissue and HepG2 cells transfected with siRNA-GAS5. Further analysis of its possible regulatory mechanism. Methods20 cases of hepatocellular carcinoma and its adjacent tissues were selected from hepatectomy,qRT-PCR was used to detect the expression of the GAS5 and Western blot was used to detect the expression of CDK6 protein. The expression levels of GAS5 in HepG2 cells were transfected with siRNA-GAS5 lipidosome. Scratch experiments,Transwell and CCK-8 were used to detect the migration,invasion and proliferation of HepG2 cells,and flow cytometry was used to detect the cell apoptosis cycle after silence. Western blot and qRT-PCR was used to validate the expression of cell cycle regulation protein Cyclin D1,CDK6 and E2 F-1. Results The expression of GAS5 in hepatocellular carcinoma tissues was significantly lower than that of adjacent tissues,the expression of CDK6 was higher than that of adjacent tissues. The migration,invasion and proliferation of HepG2 cells transfected with siRNA-GAS5 were significantly enhanced. The expression of Cyclin D1,CDK6 and E2 F-1 protein was decreased in HepG2 cells after the reduction of GAS5,the G1/S phase transition occurred after down-regulated HepG2 cells. Conclusion The expression of GAS5 is reduced in liver cancer. GAS5 can significantly inhibit hepatocellular carcinoma cells migration,invasion and proliferation. GAS5 regulate liver cancer cell cycle may be through Cyclin D1/CDK6/E2 F-1 pathway.

关 键 词：非编码RNA GAS5 肝细胞癌 细胞周期调控蛋白 

分 类 号：R349.62[医药卫生—基础医学] R604