PRL-2真核表达载体的构建及其致侵袭和粘附的研究

Construction of an eukaryotic expression vector for PRL-2 and its effect on human hepatocellular carcinoma cell invasiveness and migration in vitro

目的构建PRL-2真核表达载体并观察其致侵袭和粘附的能力。方法用RT-PCR法从肝癌细胞系HepG2总RNA中扩增编码PRL-2基因全长阅读开放框DNA,构建真核表达载体。以脂质体转染法将重组质粒稳定转染至正常永生化肝细胞系CL1中,筛选阳性克隆。分别应用RT-PCR、Westernblotting免疫印迹和免疫组化法分析PRL-2在阳性细胞中mRNA、蛋白表达及其定位。MTT法观察转染20min和120min后与底物粘附的细胞数,评价PRL-2对CL1肝细胞粘附能力的促进作用,观察6h后细胞穿透多聚碳膜上层粘蛋白的细胞数,分析PRL-2对肝细胞浸润迁移的影响。结果经RT-PCR扩增出大小为504bp的基因片断,序列测定正确并经亚克隆酶切鉴定。经过8周G418筛选后,获得稳定表达PRL-2的细胞亚系PRL-2-CL1,经RT-PCR、Western blotting印迹和免疫组化证实,PRL-2-CL1细胞系表达PRL-2mRNA及蛋白。PRL-2使CL-1细胞在20min和120min时对纤连蛋白的粘附率明显升高(P<0.05),PRL-2使CL-1细胞穿透迁移小室多聚碳膜的细胞数由(10.0±3.7)个显著升高至(44.8±2.6)个(P<0.05)。结论成功构建重组PRL-2真核表达载体并可在永生化肝细胞中稳定表达,PRL-2具有致肝癌细胞侵袭和转移的能力。

Objective To construct an eukaryotic expression vector for PRL-2 and evaluate its effect on the invasiveness and migration of a human hepatocellular carcinoma cell line. Methods RT-PCR was performed to amplify the complete PRL-2 open reading frame using the total mRNA of hepatocellular carcinoma HepG2 cells as the template. PRL-2 gene was inserted into the pGEM T easy vector and sequenced, and the correct PRL-2 sequence was subcloned into the mammalian expression vector pcDNA3.1^＋. The constructed PRL-2 vector was transfected into CL 1 cells via lipofectamine, and the stable expression of PRL-2 mRNA was detected by RT-PCR, the expressed protein identified by immunohistochemistry and Western blotting, and the effect of PRL-2 on the adhesion ability of CL-1 cell evaluated with MTT assay 20 and 120 min atter transfection. The effect of PRL-2 on the invasive migration of CL-2 cells was evaluated according to the number of cells penetrating the Matrigel layer of polycarbonate membrane of Boyden chamber. Results RT-PCR yielded a fragment of 504 bp and the inserted PRL-2 sequence was verified by sequence analysis. The subclones were identified by restriction endonuclease digestion, and a G418-resistant clone, PRL-2-CL 1, was obtained atfer 8 weeks of selection. RT-PCR showed stable expression of PRL-2 mRNA, and Western blotting confirmed overexpression of PRL-2 protein in the transfected cells. PRL-2 increased the adhesion rate of CL-1 cells to fibronectin at 20 min and 120 min atfer transfection （P〈0.05）, and also the number of CL-1 cells penetrating the polycarbonate membrane from 10.0±3.7 to 44.8±2.6 （P〈0.05）. Conclusion An eukaryotic expression vector of PRL-2 has been successfully constructed, which allows stable and efficient expression in CL-1 cell line. PRL-2 can promote cell adhesion and invasion activity of human hepatocellular carcinoma cells.