Effect of tumor suppressor gene NRPL2 mediated by CTP on apoptosis of renal cell carcinoma and activity of Bax-Caspase3 apoptotic pathway
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Effect of tumor suppressor gene NRPL2 mediated by CTP on apoptosis of renal cell carcinoma and activity of Bax-Caspase3 apoptotic pathway
Journal of Chongqing Medical UniversityVol. 40, Issue 8, Pages: 1101-1107(2015)
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重庆医科大学附属第一医院泌尿外科,重庆,400016
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Published:2015,
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Ye Mao, Tang Wei, Chang Qiyue, et al. Effect of tumor suppressor gene NRPL2 mediated by CTP on apoptosis of renal cell carcinoma and activity of Bax-Caspase3 apoptotic pathway. [J]. Journal of Chongqing Medical University 40(8):1101-1107(2015)
DOI:
Ye Mao, Tang Wei, Chang Qiyue, et al. Effect of tumor suppressor gene NRPL2 mediated by CTP on apoptosis of renal cell carcinoma and activity of Bax-Caspase3 apoptotic pathway. [J]. Journal of Chongqing Medical University 40(8):1101-1107(2015)DOI:
Effect of tumor suppressor gene NRPL2 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 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.
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