非传统型前折叠束RPB5交互因子在肝癌HepG2细胞中的生物学功能及其分子机制

Biological function and molecular mechanism of URI in HepG2 cells

目的 探讨非传统型前折叠束RPB5交互因子（URI）对肝癌HepG2细胞生物学功能的影响及其分子机制.方法 设计并构建URI真核表达和URI shRNA干扰载体,转染肝癌HepG2细胞并筛选稳定转染细胞株.采用CCK-8实验和软琼脂克隆形成实验检测各组细胞的增殖能力和体外锚定非依赖性生长的能力,采用流式细胞仪检测γ射线照射后各组细胞的细胞周期和细胞凋亡,采用Western blot法检测凋亡相关蛋白的表达量.结果 成功构建了URI表达质粒和干扰质粒,并获得相应的稳定转染细胞株.HepG2组、pCDNA3.1-URI-HepG2组和pGPU6-URIi-HepG2组细胞转染第7天相对于第1天的增殖促进率分别为（588.78±32.12）％、（959.33±58.80）％和（393.93±39.70）％,差异均有统计学意义（均P＜0.05）.HepG2组、pCDNA3.1-URI-HepG2组和pGPU6-URIi-HepG2组的克隆数分别为（43±7）个、（85±5）个和（20±4）个,差异有统计学意义（P＜0.05）.γ射线照射后,pCDNA3.1-URI-HepG2组细胞凋亡和G2/M期阻滞较HepG2组明显降低（P＜0.05）,pGPU6-URIi-HepG2组细胞凋亡和G2/M期阻滞较HepG2组明显升高（P＜0.05）.Western blot结果显示,HepG2细胞的URI、Bax和Bcl-2蛋白相对表达水平分别为0.92±0.03、1.11±0.13和0.82±0.01,pCDNA3.1-URI-HepG2组细胞的URI、Bax和Bcl-2蛋白相对表达水平分别为1.79±0.12、0.48±0.01和2.20±0.30,pGPU6-URli-HepG2组细胞的URI、Bax和Bcl-2蛋白相对表达水平分别为0.50±0.04、1.52 ±0.20和0.38±0.01.pCDNA3.1-URI-HepG2组细胞凋亡相关蛋白Bax表达水平低于HepG2组,Bcl-2蛋白表达水平高于HepG2组;pGPU6-URIi-HepG2组细胞Bax蛋白表达水平高于HepG2组,Bcl-2蛋白表达水平低于HepG2组,差异有统计学意义（P＜0.05）.结论 URI能够通过促进细胞增殖和抵抗细胞凋亡来促进肝癌细胞的生长.而干扰URI表达后,肝癌细胞的增殖能力被抑制,细胞抵抗γ射线照射的能力降低,URI可能成为肝癌治疗的新靶点。

Objective To explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor （URI） in hepatocellular carcinoma HepG2 cells.Methods The cDNA sequence and shRNA of URI were obtained and sub-cloned into eukaryotic expression vectors.Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line.The cell proliferation and anchorindependent growth in URI-overexpressing and knockdown HepG2 cells were determined by CCK-8 and soft agar colony assay.Flow cytometry was applied to detect the cell cycle and apoptosis of γ-ray irradiated cells.Apoptosis related genes were detected by Western blot.Results The pCDNA3.1-URI and pGPU6-URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained.Cell proliferation rates of the HepG2,pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were （588.78 ±32.12）%,（959.33 ±58.8）% and （393.93 ±39.7）%,respectively （P〈0.05）.The number of cell clones of HepG2,pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were 43 ± 7,85 ± 5 and 20 ± 4 （P 〈 0.05）,respectively.After γ-ray irradiation,the URI-overexpressing cell line showed a significantly lower apoptosis rate and G2/M phase arrest than those in the URI-depleted cell line （P 〈 0.05）.In the HepG2 cells,the relative protein expression levels of URI,Bax and Bcl-2 were 0.92 ± 0.03,1.11 ±0.13 and 0.82 ± 0.01 （P 〈 0.05）.In the pCDNA3.1-URI-HepG2 cells,the relative protein expression levels of URI,Bax and Bcl-2 were 1.79 ± 0.12,0.48 ± 0.01 and 2.20 ± 0.30 （P 〈 0.05）,respectively.In the pGPU6-URIi-HepG2 cells,the relative protein expression levels of URI,Bax and Bcl-2 were 0.50 ± 0.04,1.52 ± 0.20 and 0.38 ± 0.01 （P 〈 0.05）,respectively.The expression of Bax was down-regulated and Bcl-2 was up-regulated in the URI-overexpressing cell line.However,on the contrary,expression of Bax was up-regulated and Bcl-2 was down-regulated in the URI-depleted cell line.Conclusions URI may promote the growth of hepatocellular carcinoma cells via inhibition of cell proliferation and reducing the apoptosis in hepatocellular carcinoma cells in vitro.After the impairment of URI expression,the proliferation ability of hepatocellular carcinoma cells is suppressed and the ability to resist γ-ray irradiation is reduced.URI may become a potential new target for cancer therapy of hepatocellular carcinoma.