过表达钾离子通道调节蛋白抑制肝癌细胞增殖、迁移及侵袭  

Overexpression of K^+ channel regulator inhibits proliferation, migration and invasion of hepatocellular carcinoma cells

作　　者：胡啸 王艳杰 陈德[1] HU Xiao;WANG Yanjie;CHEN De(Department of Hepatobiliary Surgery, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510000, Guangdong Province, China)

机构地区：[1]广州医科大学附属第三医院肝胆外科,广东广州510000

出　　处：《肿瘤》2018年第6期535-543,共9页Tumor

摘　　要：目的:检测钾离子通道调节蛋白(K^+ channel regulator,KCNRG)在肝癌细胞中的表达情况,并探讨上调KCNRG基因表达对肝癌细胞增殖、克隆形成、迁移、侵袭和上皮-间质转化(epithelial-mesenchymal transition,EMT)能力的影响。方法:首先采用RT-PCR、实时荧光定量PCR和蛋白质印迹法分别检测人正常肝上皮永生化细胞THLE-3和肝癌细胞BEL-7402、SMMC-7721、MHCCLM3及SK-HEP1中KCNRG mRNA和蛋白的表达水平。然后采用脂质体转染法将KCNRG过表达质粒KCNRG-pc DNA3.1(+)转染至肝癌细胞SK-HEP1和MHCC-LM3中,同时设置未转染的空白对照组和转染空载体的阴性对照组。采用实时荧光定量PCR和蛋白质印迹法验证KCNRG过表达效果后,分别采用细胞计数法、克隆形成实验和Transwell小室法分别检测肝癌细胞增殖、克隆形成、迁移和侵袭能力的变化情况,并采用蛋白质印迹法检测肝癌细胞中EMT标志分子的表达变化。结果:肝癌细胞中KCNRG mRNA和蛋白表达水平明显低于人正常肝上皮永生化细胞THLE-3(P值均<0.01),尤以肝癌细胞SK-HEP1和MHCC-LM3中的表达水平最低。KCNRG过表达质粒转染后,肝癌细胞SK-HEP1和MHCC-LM3中KCNRG基因表达水平明显上调(P值均<0.01),N-钙黏蛋白(N-cadherin)表达水平明显下调(P值均<0.01),E-钙黏蛋白(E-cadherin)表达水平明显上调(P值均<0.01),并且2种肝癌细胞的增殖、克隆形成、迁移和侵袭能力均被明显抑制(P值均<0.01)。结论:肝癌细胞中KCNRG基因表达水平明显低于正常肝细胞。KCNRG基因表达上调可以抑制肝癌细胞的增殖、克隆形成、迁移、侵袭及EMT。Objective： To detect the expression level of K＋ channel regulator （KCNRG） in hepatocellular carcinoma （HCC） cells, and further to investigate the effects of up-regulation of KCNRG gene expression on the proliferation, colony formation, migration, invasion and epithelial-to-mesenchymal transition （EMT） of HCC cells.Methods： The expression levels of KCNRG mRNA and protein in human immortalized normal liver cell line THLE-3 and HCC cell lines including BEL-7402, SMMC-7721, MHCC-LM3 and SK-HEP1 were detected by RT-PCR, real-time fluorescent quantitative PCR and Western blotting, respectively. The KCNRG gene overexpression vector KCNRG-pcDNA3.1（＋） was transfected into HCC MHCC-LM3 and SK-HEP1 cells by using LipofectAMINE 2000, while the corresponding empty vector-transfection group and untransfection group were used as the negative control and blank control, respectively. The efficiency of KCNRG overexpression was verified by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the expression levels of EMT markers in MHCC-LM3 and SK-HEP1 cells with KCNRG overexpression were detected by Western blotting. The changes of proliferation, colony formation, migration and invasion abilities of the transfected MHCC-LM3 and SK-HEP1 cells were detected by cytometry, clone forming assay, Transwell chamber assay, respectively.Results： Compared with the human immortalized normal liver cell line THLE-3, the expression levels of KCNRG mRNA and protein were higher than those in HCC cells, especially in MHCC- LM3 and SK-HEP1 cells （both P 〈 0.01）. After transfection of KCNRG-pcDNA3.1（＋）, the expression ofKCNRG gene was up-regulated in MHCC-LM3 and SK-HEP1 cells （both P 〈 0.01）. The expression level of N-cadherin was significantly decreased in KCNRG gene overexpressed MHCC-LM3 and SK-HEP1 cells （both P 〈 0.01）, while the expression level of E-cadherin was significantly increased （both P 〈 0.01）. The proliferation, colony formation, migration and invasion

关 键 词：肝肿瘤 钾通道 基因表达调控 细胞增殖 细胞运动 上皮-间质转化 基因 钾离子通道调节子 

分 类 号：R735.7[医药卫生—肿瘤]