摘要
目的探索肝癌相关基因COMMD7促进HepG2细胞增殖转移的分子机制。方法设计针对COMMD7基因的特异性siR NA(small interference RNA)转染人类肝癌细胞株HepG2,作为干扰组(Si-HepG 2);并设置PTEN抑制剂处理组(Si+BpVHepG2);空白对照组(HepG2);空载体对照组(N-HepG2)。qRT-PCR检测转染后各组COMMD7、PTEN基因mR NA的表达变化;Western blot检测各组COMMD7、PTEN、p-AKT和AKT蛋白的表达变化;MSP法检测PTEN基因甲基化水平;CCK8法检测细胞增殖能力;Transwell实验检测细胞在转染前后转移及侵袭能力的改变。结果 siRNA-COMMD7转染HepG2细胞后,qR T-PCR检测结果显示,与空白对照组及空载体对照组相比较干扰组COMMD7基因的表达量降低89.9%,PTEN基因的表达量升高2.841倍,差异均有统计学意义(P<0.05);Western blot检测结果显示,siRNA干扰组COMMD7表达明显降低,PTEN表达升高;下游p-AKT表达受到显著抑制;PTEN抑制剂处理组PTEN表达受到显著抑制,下游p-AKT的表达明显增强;MSP法检测结果显示,干扰组与空白对照组及空载体对照组比较,PTEN发生明显的去甲基化,表明抑制COMMD7表达可诱导PTEN去甲基化。CKK8实验结果显示,干扰组细胞较空白对照组、空载体对照组和PTEN抑制剂处理组增殖速度明显降低,差异均有统计学意义(P<0.05);Transwell实验实验结果显示,干扰组穿膜细胞数(17.4±2.7),较空载体对照组(36.2±3.2)、空白对照组(41.6±4.5)及PTEN抑制剂处理组(47.6±1.8)明显减少,差异均有统计学意义(P<0.05)。结论 siR NA干扰下调COMMD7基因的表达,可诱导HepG2细胞内抑癌基因PTEN的去甲基化,增加PTEN蛋白的表达而抑制PI3K/AKT信号通路,以降低HepG2细胞的增殖、迁移及侵袭能力。
Objective To investigate the mechanism of COMMD7,a hepatocellular carcinoma gene,promoting human hepatocellular carcinoma cell line（ HeP G2 cells） proliferation and migration. Methods The specific siR NA（ small interference RNA） was designed for COMMD7 gene,siR NA transfected HepG 2 cells was set as Si-HeP G2 group,and the PTEN inhibitor treating group（ Si ＋ BpV-HeP G2）,the control group（ HeP G2）,the empty vector group（ HeP G2 was infected empty vector,N-HeP G2 group） were also set up. qR T-PCR was performed to evaluate the mR NA expression changes of COMMD7 and PTEN,and Western blot was performed to test the expression changes of COMMD7,PTEN,p-AKT,and AKT. MSP method was performed to detect methylation level. CCK8 was performed to test cell proliferation ability. Transwell was performed to detect the invasion of cells. Results The results of qR T-PCR showed that the expression of COMMD7 gene in Si-HeP G2 group was lower（0. 101 times） than the control group and the N-HeP G2 group,and the expression of PTEN gene was higher（2. 841 times） than the control group and the N-HeP G2 group,and all the results above were of statistically singificant difference（P 0. 05）.The results of Western blot showed that in Si-HeP G2 group,the expression of COMMD7 reduced obviously,the expression of PTEN increased and the expression of p-AKT was significantly inhibited. In Si ＋ Bpv-HeP G2 group,the expression of PTEN was significantly inhibited and the expression of p-AKT was increased. The result of MSP showed that compared with the control group and the N-HeP G2 group,Si-HeP G2 group was more obviously demethylated,which indecated that the expression of COMMD7 gene could induce PTEN demethylation. The results of CCK8 showed that the proliferation in Si-HeP G2 group was decreased compared with the control group,N-HeP G2 group and Si ＋ BpV-HeP G2 group,and the difference was singificant（ P 0. 05）. The results of Transwell showed that the numbers of cell permeating septum in SiHeP G2 group,N-HeP
出处
《局解手术学杂志》
2016年第5期313-318,共6页
Journal of Regional Anatomy and Operative Surgery
基金
国家自然科学基金(81372561)
重庆市自然科学基金(CSTC2012jjA10079)
