Effect Of Tumor Suppressor Gene NPRL2 Mediated By CTP On Apoptosis Of Renal Cell Carcinoma And Activity Of Bax-caspase3 Apoptotic Pathway

Objective: To confirm the treatment of CTP mediated tumor suppressor gene NPRL2 as CTP-NPRL2 fusing protein on human renal carcinoma cell line 786-O, and detect the apoptosis inducted and the effect of Bax-Caspase3 apoptotic pathway in renal cell carcinoma.Methods: Recombinant plasmids pET28a-CTP-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. Western blot was used to detect the protein expression of CTP-NPRL2. The CTP-NPRL2 protein and renal carcinoma cell line 786-O were co-cultured. Immunofluorescence staining and inverted fluorescence microscope were used to detect the cellular localization of CTP-NPRL2 protein in renal carcinoma 786-O. The proliferation of 786-O cells was detected by the MTT assays and the apoptosis and cell cycle were detected by flowcytometry. q RT-PCR and Western blot were used to detect the expression of Bax and Caspase-3 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 result of inverted fluorescence 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 the other two groups. q RT-PCR and Western blot suggested that, compared with the control group and NPRL2 group, the expression of Bax and Caspase-3 at the mRNA 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 inhibited their growth, possibly by affecting the activity of Bax-Caspase3 apoptotic pathway, which played a tumor suppressor role.