The Role Of CTBP2-ROCK Signal Pathway In Prostate Cancer Cell Invasion

ObjectiveTo identify the role of CTBP2level and its position transformation in regulating the biological characteristics of prostate cancer cell in vitro and preliminary discuss its molecular mechanism.MethodsThe immunohistochemical was carried out to detect the expression of CTBP2in benign prostatic hyperplasia(BPH) and prostate cancer(PCa) tissue. Westernbolt and RT-qPCR were applied to detect protein level and mRNA quantity in normal prostate cell RWPE-1and prostate cancer cell. Immunofluorescence and Laser Scanning Confocal Microscope were used to evaluate the location of CTBP2in these cells. CTBP2expressed plasmids was constructed and transformed into competent cells, which was used to amplify and extract high concentration plasmids. Then CTBP2expressed plasmids was transfected into PC3cell by liposome. G418and monoclonal cell culture were utilized to screen stable cell line, in which the mRNA quantity and protein level of CTBP2were indentified by RT-qPCR and Westernblot. The proliferation of CTBP2over expressed cells were assessed by MTT assay. Transwell was used to assess migration and invasion of CTBP2low expressed cells and CTBP2over expressed cells. Apoptosis of these two kinds of cells were evaluated by Annexin V assay. RT-qPCR was used to detect the mRNA expression of effector molecules which might be regulated by CTBP2and correlated with cell invasion. Westernblot was further used to indentify the molecules screened by RT-qPCR. All the experiment results were summarized and those need statistical analysis were analysed by SPSS statistical analysis software.ResultsCTBP2is over expressed in PCa tissue and positive relates to tumor stage and gleason score. It mainly locates in nucleus of BPH or localized PCa tissue, But appears in cytoplasm of metastatic PCa cells. Compared with normal prostate cell RWPE-1CTBP2protein level and mRNA quantity increase in PCa cell PC3. CTBP2mainly locates in nucleus in RWPE-1, while exists in nucleus and cytoplasm of PC3. We get high concentration plasmids by transforming CTBP2expressed plasmids into competent cells and amplifying plasmids in it. We then transfect CTBP2expressed plasmids into PC3through lipidosome and construct CTBP2stable ectopic over expressed cells successfully by G418screening and monoclonal cell culture, after which protein level and mRNA level of CTBP2is indentified. Cell biological behavior tests find that overexpression and mislocalization of CtBP2in PCa cell improves cell invasion significantly, however without significant effects on cell proliferation and apoptosis. While stable knockdown of CtBP2in PCa cells inhibits their invasion and increases apotosis. The results of RT-qPCR demonstrates that CTBP2knockdown suppresses mRNA expression of ROCK1in PCa cell. Westernbolt detects that protein level of ROCK1is also decreased significantly, which coordinates with the results of RT-qRCR. Protein level of p-MLC and c-Myc, which are the ROCK signaling downstream molecules, are also drop in CTBP2low expressed cells. The protein level of c-Myc downstream molecule HSPC111is also decreased.ConclusionThe abnormal expression level and location transform of CTBP2in PCa cells impacts on its biological behavior in vitro. CTBP2-ROCK signaling pathway may be exist in PCa cell, which may be one of the molecular mechanisms to promote PCa cell invasion.