GPX4对肾透明细胞癌增殖转移能力的影响及其与IGF-1R、COX-2表达的关系

Effect of GPX4 on proliferation and metastasis of renal clear cell carcinoma and its relationship with expression of IGF-1R and COX-2

目的 探究人谷胱甘肽过氧化酶4(GPX4)对肾透明细胞癌增殖转移能力的影响及其与IGF-1R、COX-2表达的关系.方法 体外培养人正常肾小管细胞系HK-2及人肾透明细胞癌细胞Caki-1、A498、Caki-2、786-o,即时荧光定量PCR技术(RT-PCR)及蛋白免疫印迹实验(Western blot)检测不同细胞系中GPX4 mRNA及蛋白表达,通过慢病毒转染法构建过表达GPX4细胞系,包括:空白载体组及GPX4过表达组,及干扰GPX4肾透明细胞癌细胞系,包括:对照序列组、GPX4干扰1组及GPX4干扰2组,RT-PCR检测各组细胞GPX4、IGF-1R及COX-2 mRNA表达水平,Western blot检测各组细胞GPX4、IGF-1R及COX-2蛋白表达水平,CCK-8实验检测各组细胞0、24、48、72及96 h相对增殖能力,Transwell侵袭及迁移实验检测各组细胞侵袭及迁移能力.结果 相比人正常肾小管细胞系, GPX4在肾透明细胞癌细胞系中高表达;GPX4过表达组细胞相比空白载体组细胞GPX4、IGF-1R及COX-2 mRNA及蛋白表达显著增高,GPX4干扰1组及GPX4干扰2组细胞相比对照序列组细胞GPX4、IGF-1R及COX-2 mRNA及蛋白表达显著降低;GPX4过表达组细胞相比空白载体组细胞72及96 h增殖能力显著增高,GPX4干扰1组及GPX4干扰2组细胞相比对照序列组细胞72及96 h增殖能力显著降低;GPX4过表达组细胞侵袭及迁移细胞数显著高于空白载体组细胞,GPX4干扰1组、GPX4干扰2组细胞侵袭及迁移细胞数显著低于对照序列组细胞.结论 GPX4在肾透明细胞癌细胞中高表达,与IGF-1R及COX-2的表达正相关,并可促进细胞体外增殖及转移能力.

Objective To investigate the effect of human glutathione peroxidase 4(GPX4)on the proliferation and metastasis of renal clear cell carcinoma and its relationship with the expression of IGF-1R and COX-2.Methods Culture of human normal tubular cell line HK-2 and human renal clear cell carcinoma Caki-1,A498,Caki-2,786-o in vitro.Detection of GPX4 mRNA and protein expression in different cell lines by quantitative real-time PCR(RT-PCR)and Western blot assay.Overexpression of GPX4 cell lines,including blank carrier(Vector)and overexpress GPX4(oeGPX4)group,and interference with GPX4 renal clear cell carcinoma cell lines,including random sequence(shControl),interference GPX4#1(shGPX4#1)and interference GPX4#2(shGPX4#2)group by lentiviral transfection.RT-PCR technology and Western blot were used to detect the expression of GPX4,IGF-1R and COX-2 mRNA and protein.CCK-8 assay was used to detect the relative proliferation of cells at 0,24,48,72 and 96 h in each group.Transwell invasion and migration assay to detect the invasion and migration ability of cells of each group.Results GPX4 is highly expressed in renal clear cell carcinoma cell lines compared to human normal tubular cell lines;The expression of GPX4,IGF-1R and COX-2 mRNA was significantly increased in oeGPX4 cells compared with Vector cells,the expression of GPX4,IGF-1R and COX-2 mRNA was significantly decreased in shGPX4#1 and shGPX4#2 compared with shControl cells;oeGPX4 cells significantly increased proliferative capacity compared to Vector cells at 72 and 96 h,the proliferation of shGPX4#1 and shGPX4#2 cells was significantly lower than that of shControl cells at 72 and 96 h;The number of invading and migrating cells of oeGPX4 cells was significantly higher than that of Vector cells,the number of invasive and migrating cells in shGPX4#1 and shGPX4#2 cells was significantly lower than that in shControl cells.Conclusion GPX4 is highly expressed in renal clear cell carcinoma cells,which is positively correlated with the expression of IGF-1R and COX-2,and can promote cell proliferation and metastasis in vitro.