TAZ/WWTR1影响肝癌细胞SMMC-7721的侵袭、迁移能力

The Effect of TAZ/WWTR1 on Migration and Invasion of SMMC-7721 Cells

目的探讨转录共激活子TAZ/WWTR1对肝癌细胞株SMMC-7721侵袭、迁移的影响,为寻找原发性肝细胞癌的防治提供理论基础。方法 TAZ基因靶向干扰RNA的设计合成:从TAZ的信使RNA(mRNA)中挑选出合适的siRNA靶点序列作为候选,序列通过基因BLAST,得出三对siRNA;TAZ过表达质粒的构建:从SMMC-7721细胞中提取TAZ基因,使用pCMV5-HA为载体构建人TAZ过表达质粒;细胞转染:以Lipofectamin^(TM)2000为载体,将TAZ siRNAs转入SMMC-7721细胞中,应用实时RT—PCR和Western blotting检测TAZ的表达情况,筛选出干扰效果最佳的siRNA,并进一步比较转染了TAZ siRNA组与转染了阴性对照组的SMMC-7721细胞侵袭、迁移能力的变化;构建稳定过表达TAZ基因的人肝癌细胞(SMMC-7721)系:用Lipofectamin^(TM)2000将TAZ过表达质粒转染入SMMC-7721细胞中,使用G418筛选出稳定高表达TAZ的细胞系,应用Transwell侵袭实验观测TAZ稳定过表达的SMMC-7721细胞的侵袭、迁移能力。结果三对siRNA均可使TAZ表达下调,其中siRNA-1效果最佳;与阴性对照组相比,转染了TAZ siRNA-1的SMMC-7721细胞的侵袭、迁移能力下降;经过鉴定确定为稳定过表达TAZ的SMMC-7721细胞的侵袭、迁移能力相比于阴性对照组显著升高。结论本研究通过靶向干扰和稳定过表达TAZ基因的方法证实TAZ能够影响人肝癌细胞株SMMC-7721的侵袭、迁移能力,为揭示原发性肝癌恶性转移的发病甚至启动机制提供一个崭新思路。

Objective To investigate the effect of transcriptional coactivator TAZ/WWTR1 on invasion and migration of hepatocellular carcinoma cell line SMMC-7721 and to provide a theoretical basis for prevention and treatment scheme of primary hepatocellular carcinoma. Method The design and synthesis of TAZ targeted small interference RNA （siRNA）： the suitable siRNA target sequence selected from the messenger RNA （mRNA） of TAZ were regarded as candidates. With gene BLAST, three siRNAs were selected..The construction of the over- expression plasmid of TAZ： TAZ gene was taken from the SMMC-7721 cells, then used the pCMV5-HA vector to construct TAZ plasmid. Cell transfection： TAZ siRNAs were transfected into SMMC-7721 cells by using LipofectaminTM2000, the expression of TAZ were detected by real-time quantitative polymerase chain reaction and Western blotting. Then we transfected the most effective siRNA into SMMC-7721 cells and compared the cells’ ability of migration and invasion with cells which were transfected negative control siRNA. The construction of TAZ stable high expression cell line： TAZ over expression plasmid were transfected into SMMC-7721 cells, G418 were used to select the TAZ stable high expression colonies. The cell migration and invasion assay were used to observe the TAZ stable high expression SMMC-7721 cell line. Results All of three siRNA could down regulate the expression of TAZ, and the siRNA-1 was the most effective. Comparing with the negative control group, the group which were transfected TAZ siRNA-1 had a low ability of migration and invasion. However, the defined TAZ stable high expression SMMC-7721 cell line got an increased migration and invasion ability. Conclusions In this study, we used targeted siRNA and stable over expression TAZ cell line to demonstrate that TAZ can affect the human hepatocellular carcinoma cell line SMMC-7721 ’s invasion and migration ability. This finding provides a new idea to reveal the mechanism of hepatocellular carcinoma’s metastasis and possible carcinogensis process.