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

摘要:

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pET28a-CTP-NPRL2和pET28a-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1.0 μmol/L CTP-NPRL2蛋白、2.0 μmol/L CTP-NPRL2蛋白、4.0 μmol/L CTP-NPRL2蛋白、4.0 μmol/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mRNA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mRNA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。

Abstract:

Objective：To confirm the treatment of CTP mediated tumor suppressor gene NRPL2 as CTP-NPRL2 fusing protein on hu－man 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 pET28a-CTP-NPRL2 and pET28a-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-cul－tured 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 pro－liferation 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 mRNA 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 import－ing 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 sug－gested that，compared with those of the control group and NPRL2 group，the expression of Bax and Caspase-3 at the mRNA and pro－tein 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 path－way，thus play a tumor suppressor role.