Objective:The aim of our study was to investigate the effects of Pin1 reduction on SW620 cell proliferation and apoptosis in human colorectal carcinoma.Methods:We constructed a plasmid of RNA interfering(shRNA) for Pin1 gene(pGenesl-1-Pin1),then the plasmid was transfected into colorectal carcinoma SW620 cells line by liposome mediation.The protein expression of Pin1 was tested by Western blotting.The proliferation rate was analyzed by MTT and the apoptotic rate of cells was tested by flow cytometry.In order to explain further the effect of Pin1 in SW620 cells,the protein level of Bcl-2 was analyzed by Western blotting.Results:pGenesil-1-Pin1 plasmid was successfully constructed and confirmed by sequencing.The protein relative levels of Pin1 were 0.06 ± 0.04 for the P-shRNA/SW620 cells,and 0.32 ± 0.09 for the P-Con/SW620 cells.The cell growth rate of SW620 cells was slower while the apoptotic rate was increased after transfection with pGenesil-Pin1 plasmid,and the apoptotic rate was 12.38% ± 1.55% for the P-shRNA/SW620 group.At the same time,we found that the protein expression of Bcl-2 was also reduced.The results were 0.13 ± 0.04 for the P-shRNA/SW620 cells,and 0.36 ± 0.08 for the P-Con/SW620 cells.Conclusion:Inhibited Pin1 expression may suppress the cell proliferation and promote apoptosis of colorectal carcinoma cells in vitro.
Yan Zhu Liyuan Qin Meining Li Dong Zhang Yuehong Zhang Niuliang Cheng
Objective： The aim of our study was to investigate the effect of Pin 1 on the telomerase activity in human colorectal carcinoma HCT116 cells. Methods： Firstly, we transfected plasmid pGenesil-l-Pinl （p-shRNA） using liposome （Lipofectamine 2000） into colorectal cancer HCT-116 cells to down-regulate the expression of Pin1. To detect the apoptotic rate of HCT116 cells was by cytometry （FCM）. The expression of Pin1 and hTERT at RNA levels in human colorectal cancer HCT116 cells were determined by RT-PCR. To evaluate the activity of telomerase was by TRAP-silver staining. The subcellular localization and accumulative level of p-NF-κB/p65 protein at the nuclear was detected by Immunofluorescence and Western blotting. The DNA-binding activity of NF-κB/p65 was detected by electrophoretic mobility shift assay （EMSA）. Results： Using liposome into colorectal cancer HCT-116 cells, and down-regulate the expression of Pin1 （0.392 ± 0.072-fold; P = 0.001）, and the apoptotic rate was increased （11.40% ± 1.54%; P 〈 0.05）. Compared with transfected p-CON cell group, in transfected p-shRNA cell group, the transcription of hTERT was lower （0.171 ±0.060-fold; P = 0.001） by quantitative real-time RT-PCR, and the results of TRAP-silver staining analysis suggested that the telomerase activity was significantly declined （0.384 ± 0.015-fold; P 〈 0.05）. Furthermore, it was demonstrated by Immunofluorescence that p-NF-κB/p65 had a nuclear localization, and the level of p-NF-κB/p65 protein at the nuclear was reduced with silencing the expression of Pin1 by Western blotting. Using EMSA, it was suggested that NF-κB/p65 was able to bind to hTERT promoter, and the direct interaction was declined with silencing the expression of Pin1. Conclusion： Taken together, silencing Pin1 may suppress activity of telomerase and the expression of hTERT by inhibiting NF-κB/p65 activity and reducing the combination of NF-κB/p65 and hTERT gene promoter.
