胞浆转导肽介导抑癌基因NPRL2对肾癌细胞凋亡及Bax-Caspase3凋亡通路的影响

Effect of tumor suppressor gene NRPL2 mediated by CTP on apoptosis of renal cell carcinoma and activity of Bax-Caspase3 apoptotic pathway

目的:证实胞浆转导肽(cytoplasmic transduction peptide,CTP)介导抑癌基因NPRL2以CTP-NPRL2融合蛋白形式对人肾癌细胞株786-O的作用,检测其诱导的肾癌细胞凋亡及其对Bax-Caspase3凋亡通路的影响。方法:构建重组质粒p ET28aCTP-NPRL2和p ET28a-NPRL2并转染大肠杆菌DH5α,从而得到原核表达体系用于CTP-NPRL2融合蛋白及NPRL2蛋白的表达和提取;Western blot验证CTP-NPRL2蛋白和NPRL2蛋白的表达;将肾癌786-O细胞分为2组,分别与等量的NPRL2蛋白和CTP-NPRL2蛋白共培养,采用免疫荧光染色和激光共聚焦显微镜检测2种蛋白在肾癌细胞786-O的亚细胞定位情况;将在96孔板培养的786-O细胞分为5组,其中4组分别用1.0μmol/L CTP-NPRL2蛋白、2.0μmol/L CTP-NPRL2蛋白、4.0μmol/L CTP-NPRL2蛋白、4.0μmol/L NRPL2蛋白处理,剩余1组为对照组;MTT法检测各组786-O细胞增殖的情况;流式细胞仪分析各组细胞周期和细胞凋亡情况;real-time PCR、Western blot检测各组786-O细胞中Bax和Caspase-3在m RNA及蛋白水平的表达情况。结果:质粒测序结果显示成功构建所需的原核表达载体;Western blot结果显示CTP-NPRL2融合蛋白在原核表达载体中有表达。激光共聚焦显微镜结果提示CTP-NPRL2蛋白导入786-O细胞后定位于胞浆;MTT发现CTP-NPRL2组细胞增殖较NPRL2组及空白对照组受到明显的抑制(P<0.05);流式细胞分析显示,相对于空白对照组和NPRL2组,CTP-NPRL2组的凋亡率显著增高且处于G0/G1期细胞明显增多(P<0.05)。real-time PCR和Western blot结果提示,与空白对照组和NPRL2组比较,CTP-NPRL2组Bax和Caspase-3在m RNA和蛋白质水平的表达均明显上调(P<0.05)。结论:CTP介导抑癌基因NRPL2以CTP-NPRL2融合蛋白形式导入肾癌786-O细胞中并对其有明显的抑制作用,可能通过影响Bax-Caspase3凋亡通路的活性,抑制786-O细胞的增殖,并诱导其凋亡将细胞周期阻滞于G0/G1期,从而发挥抑癌作用。

Objective：To confirm the treatment of CTP mediated tumor suppressor gene NRPL2 as CTP-NPRL2 fusing protein on human renal carcinoma cell line 786-O,and to detect the apoptosis of renal cell carcinoma and activity of Bax-Caspase3 apoptotic pathway. Methods：Recombinant plasmids p ET28a-CTP-NPRL2 and p ET28a-NPRL2 were constructed and then transfected into E.coli DH5α,then the prokaryotic expression systems were got to express and collect CTP-NPRL2 fusing protein and NPRL2 protein. Western blot was used to detect the protein expression of CTP-NPRL2 and NPRL2. The 786-O cells were divided into 2 groups and co-cultured with an equal amount of NPRL2 protein and CTP-NPRL2 protein,respectively. Immunofluorescence staining and laser scanning confocal microscopy were used to detect the subcellular localization of the proteins in 786-O cells. The 786-O cells cultured in 96-well plates were divided into 5 groups,4 groups of all were treated with 1.0 μmol/L CTP-NPRL2 protein,2.0 μmol/L CTP-NPRL2 protein,4.0 μmol/L CTP-NPRL2 protein and 4.0 μmol/L NRPL2 protein. The remaining group was taken as a control group. The proliferation of 786-O cells in each group was detected by the MTT assays,the apoptosis and cell cycle in each group were detected by flowcytometry. Real-time PCR and Western blot were used to detect the expression of Bax and Caspase-3 in each group at the levels of m RNA and protein,respectively. Results：The plasmid sequencing showed that the prokaryotic expression vectors were constructed successfully. Western blot showed CTP-NPRL2 fusing proteins were expressed in the prokaryotic expression vectors. The results of laser scanning confocal microscope pointed CTP-NPRL2 proteins were localized in the cytoplasm after importing into 786-O cells. MTT assay indicated that compared with the other two groups,CTP-NPRL2 group showed a significant decrease in proliferation（P〈0.05）. Flowcytometry assay demonstrated that the apoptotic rate and the proportion of the cells at G0/G1 phase were both increased（P〈0.05）in CTP-NPRL2 group compared with those of the other two groups. Real-time PCR and Western blot suggested that,compared with those of the control group and NPRL2 group,the expression of Bax and Caspase-3 at the m RNA and protein levels in CTP-NPRL2 group were significantly increased（P〈0.05）. Conclusion：CTP-NPRL2 fusing proteins could import into renal carcinoma cell 786-O and significantly inhibit their growth,possibly by affecting the activity of Bax-Caspase3 apoptotic pathway,thus play a tumor suppressor role.