Ezrin上调HepG2细胞膜转运蛋白MRP2的表达

Ezrin knockdown up-regulates MRP2 protein membrane expression in HepG2 cells

目的建立Ezrin基因表达缺失的Hep G2细胞株,并研究其在胆汁淤积时对Hep G2细胞胆汁酸转运蛋白多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)的影响。方法不同位点干扰的Ezrin-shRNA质粒转染Hep G2细胞,筛选Ezrin基因表达缺失的Hep G2细胞。分别提取细胞总蛋白和膜蛋白,检测Ezrin基因表达缺失是否影响Hep G2细胞胆汁酸转运蛋白MRP2的表达。结果 RT-q PCR结果证实,Ezrin-shRNA#3质粒转染Hep G2细胞后成功抑制了Ezrin基因表达,抑制率为70%。蛋白免疫印迹表明,Ezrin基因表达缺失后,Hep G2细胞胆汁酸转运蛋白MRP2表达增高,但与对照组相比,差异无统计学意义(P>0.05)。蛋白免疫共沉淀检测生物素化MRP2膜蛋白表明,Ezrin基因表达缺失后,无论是胞浆中还是细胞膜上,MRP2膜蛋白均表达增高(P<0.05)。结论 Ezrin基因表达缺失后能够上调Hep G2细胞中MRP2膜蛋白,使其表达增高。

Objective To determine the role of ezrin in the regulation of multidrug resistanceassociated protein 2( MRP2) by constructing ezrin knockdown Hep G2 cell lines through stably transfected with Ezrin-shRNA plasmids. Methods Ezrin-shRNA plasmids targeting to silence the gene at different sites were used to transfect Hep G2 cells. Then Hep G2 cell lines stably transfected by Ezrin-shRNA plasmids were obtained by G418 selection. The knockdown efficiency was measured by real-time q PCR and Western blotting.The expression of membrane transport protein MRP2 was detected in both total membrane extraction and biotinylated cell surface proteins to explore the effect of ezrin knockdown. Results Real-time q PCR results revealed that the transfection of Ezrin-shRNA#3 plasmid reduced the ezrin expression,with an inhibitory rate of about 30% of control cells( P < 0. 05). Western blotting indicated that ezrin knockdown resulted in the increased expression of MRP2,but no such increase was seen in the control cell though without significant difference( P > 0. 05). Immunoprecipitation assay showed ezrin knockdown induced the expression of MRP2 protein increased in both total membrane extraction and biotinylated cell surface proteins( P < 0. 05).Conclusion Ezrin knockdown induces MRP2 protein membrane expression by post-translational regulation in Hep G2 cells.