整合蛋白β_1 shRNA表达质粒的构建及其对肝癌细胞SMMC-7721增殖的影响

Construction of integrin β_1 shRNA expression plasmid and its regulation on proliferation in cell line SMMC-7721

目的应用RNAi技术,构建整合蛋白β1 shRNA表达质粒,诱导肝癌细胞中整合蛋白β1基因沉默,并观察其对肝癌细胞SMMC-7721增殖的影响。方法设计整合蛋白β1 shRNA序列,与载体pSilencer 2.0连接,构建整合蛋白β1 shRNA表达质粒。经测序鉴定后,转染肝癌细胞SMMC-7721,Western blot检测整合蛋白β1及p27的蛋白表达,MTT方法测定转染后细胞的增殖能力。结果成功构建了整合蛋白β1 shRNA表达质粒,转染肝癌细胞SMMC-7721后,整合蛋白β1的蛋白表达被明显抑制,p27的蛋白表达也随之减少。MTT实验结果发现转染细胞的增殖能力明显提高。结论应用RNAi技术,可特异性阻断整合蛋白β1的蛋白表达。整合蛋白β1可调节肝癌细胞的增殖,并可能与p27的表达相关。

Objective To construct integrinβ1 subunit small interfering RNAs expression plasmid and to observe its influence on the proliferation in human cell line SMMC-7721. Methods Using RNA interference techniques, a DNA vector-driven shRNAs expression plasmid was constructed with pSilencer2.0 vector containing 136 promotor. After sequencing, this plasmid was transfected into SMMC-7721. Then the transfected cells were harvested, and the expression of integrinβ1 and p27 protein were detected by western blot. Meanwhile, the changes of cell proliferation were analyzed by MTT assay. Results The integrinβ1 shRNA expression plasmid was constructed successfully. After plasmid transfection, the protein amount of both integrin β1 and p27 were reduced. Moreover, MTT assay indicated that the proliferation ability of transfected cells was enhanced compared with the negative control group. Conclusion shRNA expression plasmid could specifically inhibit the protein expression of integrinβ1. Integrinβ1 could regulate the cell growth in SMMC-7721, and p27 may play an important role herein.