shRNA沉默Kir6．2基因表达载体的构建及其对HepG2细胞增殖和侵袭的影响

Construction of shRNA expression vector silencing Kir6.2 gene and to study its influence on the proliferation and invasion of HepG2 cells

目的构建shRNA体内表达载体pGenesil-3-shRNA，研究抑制Kir6．2基因表达对HepG2细胞增殖和侵袭的影响。方法以Kir6．2基因为目的基因，以产生shRNA质粒载体pGenesil-3为表达模板，细胞内转录合成2条shRNA。重组质粒经BamH Ⅰ和Hind Ⅲ酶切鉴定和测序后，使用Lipofectamine 2000转染HepG2细胞。实验分为SK组（未转染）、SK-HK组（阴性对照）、SK-K1（转染干扰序列1）组和SK-K2（转染干扰序列2）组。G418筛选出荧光单克隆进行培养。Western blot检测Kir6．2蛋白在各组细胞中的表达。MTT法和Transwell系统分别检测各组细胞的增殖和侵袭能力。结果经酶切、测序鉴定，重组质粒符合设计要求。Western blot检测结果显示SK组、SK-HK组、SK-K1组和SK-K2组Kir6．2蛋白质相对表达量分别为0．9349±0．0443，0．9098±0．0195，0．2869±0．0295，0．3256±0．0341；和对照组相比，实验组Kir6．2蛋白减少。MTT法检测SK组、SK-HK组、SK-K1组、SK-K2组细胞的增殖分数分别为0．7523±0．0681，0．7351±0．0589，0．2494±0．0485，0．2261±0．0298，F=0．2401，P=0．000。Transwell实验结果显示，SK组、SK-HK组、SK-K1组、SK-K2组细胞的穿膜数分别为47．0±7．1，40．7±8．0，14．3±5．1，15．0±3．7，F=30．74，P=0．000。和对照组相比，实验组HepG2细胞增殖和侵袭能力均明显降低。结论抑制HepG2细胞Kir6．2基因的表达可降低HepG2细胞的增殖和侵袭能力。

Objective To construct the expression vector pGenesil-3-shRNA that can express the short hairpin RNAs （shRNA） silencing Kir6.2 gene and to study its influence on the proliferation and invasion of HepG2 cells. Methods Two shRNA silencing Kir6.2 genes were transcript synthesized intracellularly by expressed templates of plasmid vector pSilence-3, and the target sequence of Kit6.2 gene was inserted into the upstream of the reporter gene in order to construct the recombinant plasmid vector pGenesil-3. Plasmids containing 2 different sequences of human Kir6.2 mRNA coding region were constructed and transfected into HepG2 cells by using lipofectamine 2000 methods. The experiment was divided into 4 groups： SK （normal）, SK-HK （negative control）, SK-K1 （transfected with the interfering sequence 1） and SK-K2 （transfected with the interfering sequence 2） groups. A selected single clone was cultured after screening by G418. The expression of Kir6.2 protein was detected by Western blot. MTT assay and Transwell system were used to observe the proliferation and invasion of HepG2 cells. Results The recombinant expression plasmid pGenesil-3 was successfully constructed and underexpression of Kir6.2 gene in HepG2 cells was detected by Western blot. Underexpression of Kir6.2 gene significantly decreased the proliferation and invasion of the HepG2 cells. Conclusion shRNA can inhibit the expression of Kir6.2 gene in the HepG2 cells, and underexpression of Kir6.2 gene decreased the proliferation and invasion of the HepG2 cells.