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

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.