小鼠HuR真核表达载体构建及对Lewis肺癌细胞增殖和侵袭的影响

Construction of human antigen R eukaryotic expression vector and influence of HuR expression on the proliferation and invasion of Lewis lung cancer cells

目的构建小鼠同源的人抗原R(HuR)基因真核表达载体,观察HuR过表达对Lewis肺癌(LLC)细胞增殖和侵袭的影响。方法从LLC细胞中抽提总RNA,RT-PCR扩增HuR cDNA,基因工程方法将PCR片断克隆至真核表达载体pEGFP-N1的多克隆位点。重组质粒经EcoRI和SmaI双酶切鉴定和测序后,使用LipofectamineTM2000转染LLC细胞。Western blot-ting检测HuR在细胞中的表达。MTT和Transwell系统分别检测细胞增殖和侵袭能力。结果经酶切、测序鉴定,小鼠HuR基因正确插入到pEGFP-N1的多克隆位点,获得pEGFP-HuR;Western blotting显示转染组与未转染组比较,HuR蛋白表达、LLC细胞增殖能力和侵袭能力均显著增强。结论HuR过表达可提高肿瘤细胞的增殖和侵袭能力。

Objective To construct a eukaryotic expression vector containing human antigen R gene, and observe the effects of HuR overexpression on the proliferation and invasion of mouse Lewis lung cancer （LLC） cells. Methods Total RNA from LLLC cells was obtained by Tripure reagent. Mouse HuR cDNA was amplified by RT-PCR and cloned into pEGFP-N1 vector using gene engineering technology. The recombined expression vector pEGFP-HuR was confirmed by restriction digestion with EcoRI and SmaI and sequencing, and then transfected to LLC cells by Lipofectamine^TM 2000 reagent. Expression of HuR protein was determined by Western blotting. MTT assay and transwell system were used to observe the growth and invasion of LLC cells, respectively. Results The recombinant expression plasmid pEGFP-HuR was successfully constructed and overexpression of HuR gene in LLC cells could be detected by Western blotting. Overexpression of HuR increased significantly cell proliferation and invasion. Conclusion Overexpression of HuR enhances tumor cell proliferation and invasion.