COMMD7基因通过细胞外调节蛋白激酶/丝裂原活化蛋白激酶信号通路促进肝癌细胞HepG2增殖的研究  

COMMD7 gene promotes HepG2 proliferation via extracellular regulated protein kinase/mitogen-activated protein kinase signaling pathway

作　　者：商阳阳 郑璐[1] 尤楠[1] 黄小兵[1] 顾勋鑫 李靖[1] 

机构地区：[1]第三军医大学新桥医院肝胆外科,重庆400037

出　　处：《中华消化外科杂志》2015年第4期316-320,共5页Chinese Journal of Digestive Surgery

基　　金：国家自然科学基金（81372561）;重庆市自然科学基金（CSTC2012jjA10079）

摘　　要：目的 采用RNA干扰沉默COMMD7基因,观察人肝癌细胞HepG2的变化,探讨其相关作用机制.方法 设计COMMD7基因的干扰RNA片段（shRNA）,构建COMMD7-shRNA质粒.将肝癌细胞HepG2分为3组：空白组不做转染,control-shRNA组转染空载体,COMMD7-shRNA组转染阳性载体.转染结束后在荧光显微镜下观察细胞形态.采用Western blot检测COMMD7蛋白的表达.采用CCK-8检测细胞活力,流式细胞仪检测细胞凋亡.采用Western blot检测细胞外调节蛋白激酶（ERK）/丝裂原活化蛋白激酶（MAPK）信号通路中的下游分子ERK1/2和MEK1/2蛋白的表达及其磷酸化水平.符合正态分布的计量资料以x^-±s表示,多组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果 荧光显微镜下转染成功的细胞为椭圆形或梭形,呈绿色荧光.Western blot检测结果显示：空白组、control-shRNA组和COMMD7-shRNA组肝癌细胞HepG2中COMMD7蛋白的相对表达量分别为0.90±0.18、1.03±0.05和0.23 ±0.03,3组比较,差异有统计学意义（F=152.08,P＜0.05）;COMMD7-shRNA组COMMD7蛋白相对表达量低于空白组和control-shRNA组,两组比较,差异有统计学意义（t=20.74,21.16,P＜0.05）.CCK-8检测结果显示：空白组、control-shRNA组和COMMD7-shRNA组肝癌细胞HepG2细胞活力分别为1.193±0.024、1.225±0.034和1.147 ±0.021,3组比较,差异有统计学意义（F=6.90,P＜0.05）;COMMD7-shRNA组肝癌细胞HepG2细胞活力低于空白组和control-shRNA组,两组比较,差异有统计学意义（t=3.53,3.69,P＜0.05）.流式细胞仪检测结果显示：空白组、control-shRNA组和COMMD7-shRNA组肝癌细胞HepG2凋亡率分别为6.1％±0.3％、7.8％±0.5％和20.9％±1.4％,3组比较,差异有统计学意义（F=270.80,P＜0.05）;COMMD7-shRNA组肝癌细胞HepG2凋亡率高于空白组和control-shRNA组,两组比较,差异有统计学意义（=21.77,19.36,P＜0.05）.Western blot检测结果显示：空白组、control-shRNA组和COMMD7-shRNA组肝癌细胞HObjective To observe the changes of the cells of human hepatocellular carcinoma （HepG2）using RNA for silencing the expression of COMMD7 gene,and investigate related mechanism of COMMD7 gene promoting HepG2 proliferation.Methods COMMD7 gene shRNA was designed and constructed into COMMD7-shRNA plasmid.HepG2 cells were divided into the HepG2 group,control-shRNA group （empty vectors were infected） and COMMD7-shRNA group （positive vectors were infected）.Cells shapes were observed by fluorescence microscope after infecting.The expression of COMMD7 and expression and phosphosylation of extracellular regulated protein kinase1/2 （ERK1/2） and MEK1/2 protein were measured by Western blot.The cell vitality was measured by cholecystokinin octapeptide （CCK-8）,and the apoptosis of cell was detected by flow cytometry.The measurement data with normal distribution were presented as x^- ± s.The comparisons among groups were evaluated with the one-way ANOVA,and pairwise comparison was analyzed by the LSD-t test.Results The cells were oval or spindle shapes and displayed green fluorescent after infected successfully.The results of Western blot showed that the relative quantitative expression of COMMD7 protein in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.90 ±0.18,1.03 ±0.05 and 0.23 ±0.03,respectively,with a significant difference among the 3 groups （F =152.08,P 〈 0.05）,and the expression of COMMD7 protein in the COMMD7-shRNA group was significantly lower than those in the other 2 groups （t =20.74,21.16,P 〈 0.05）.The results of CCK-8 showed that the scores of the HepG2 vitality in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 1.193 ±0.024,1.225 ±0.034 and 1.147 ±0.021,respectively,with a significant difference among the 3 groups （F =6.90,P 〈 0.05）,and the HepG2 vitality in the COMMD7-shRNA group was significantly lower than those in the other 2 groups （t =3.53,3.69,P 〈 0.05）.The results of flow cytometry showed that the apoptosis rate of HepG2

关 键 词：肝肿瘤 HEPG2细胞 COMMD7基因 细胞外调节蛋白激酶 丝裂原活化蛋白激酶 

分 类 号：R735.7[医药卫生—肿瘤]