The Effect Of CtBP2 On The Biological Behavior Of Prostate Cancer Cell

Objective:In this study, we established C-terminal-binding protein 2(Ctbp2) knockout prostate cancer cell C4-2 line by Ct BP2-sh RNA to investigate the effect of Ctbp2 downregulation on the C4-2 cell proliferation in vitro and tumor growth in vivo, and the effect of Ctbp2 downregulation on the C4-2 cell apoptosis. Moreover, we investigate the effect of Ctbp2 downregulation on the C4-2 cell invasion and migration ability in vitro and in vivo, and attempted to elucidate the mechanism of how Ctbp2 affect the invasion and migration ability of C4-2 cell. Methods:1. The expression of Ct BP2 m RNA in 60 prostate cancer tissues and 60 adjacent normal prostate tissues were assessed by Real-time florescent quantitative polymerase chain reaction(RT-q PCR). Human prostate cancer C4-2 cells were infected with lentivirus carrying short hairpin RNA(sh RNA) targeting Ctbp2 expression or the negative control plasmids. The expression of Ctbp2 m RNA and protein were assessed by RT-q PCR and western blot to verify the transfection efficiency, respectively. The effect of Ctbp2 downregulation on the proliferation and apoptosis of C4-2 cells in vitro were detected by cell counting kit-8(CCK-8) assay and flow cytometry, respectively. The effect of Ctbp2 downregulation on the proliferation of C4-2 cells in vivo was invested by nude mouse. The effects of Ctbp2 specific substrate expourse on the proliferation of C4-2 cell was detected by CCK-8 assay. The effects of Ctbp2 downregulation on the expression of apoptosis-related protein BIK was assessed by western blot. 2. The effects of Ctbp2 downregulation on invation and migration of C4-2 cell in vitro and in vivo were assessed by Transwell assay and small animals living imaging technology, respectively. The expression level of E-cadherin m RNA was detected by RT-q PCR and the expression level of E-cadherin and N-cadherin protein were detected by western blot. Co-IP assay was used to explore the relationship between Ctbp2 and Slμg. Phalloidine staining was used to detect the cytoskeletal structure changes of C4-2 cell followed by Ct BP2 downregulation. We searched the information of the relationship between Ct BP2 and Rho C in the UCSC database. Rho C m RNA and proteins of Rho C, ROCK1, P-MLC expression levels followed by Ct BP2 downregulation were detected. Results:1. The expression level of Ct BP2 m RNA in human prostate cancer tissues was significantly higher than in the adjacent normal prostate tissues(1.00±0.41 vs 2.79±1.04)(P<0.05). By immuno-histochemical staining, we found that Ctbp2 protein was highly expressed in huaman prostate cancer tissue. Stable Ctbp2-sh RNA transfection significantly down-regulated the expression of m RNA and protein levels of Ctbp2 when compared with vector group cells. Ctbp2 downregulation could significantly decrease the proliferation capability of C4-2 cells in 24 h when compared with control group(0.34±0.05 vs 0.59±0.11), meanwhile there was no significantly difference between vector group and control group cells(0.58±0.09 vs 0.59±0.11). The growth rate of xenograft tumor formed by C4-2 cell in nude mice was also significantly inhibited by Ctbp2 knockdown compared with vector group(893.28±241.15 mm3 vs 2173.83±472.71 mm3)(P<0.05). The cell clone forming experiments showed that downregulate Ctbp2 expression could inhibite clone formation of C4-2 cell. The proliferation rate of C4-2 cell could be suppressed by Ct BP special substrate MTOB, and positive correlations with the concentration of MTOB. Cell apoptosis assay was carried out by flow cytometry, and the result showed that compared with cont group cells downregulate Ctbp2 expression significantly increased the apoptosis rate of C4-2 cells(41.00%±3.01% vs 10.10%±0.96%)(P<0.05). The apoptosis-related protein BIK expression level was significantly elevated post Ctbp2 knocked out in C4-2 cells. 2. The effect of Ct BP2 on the invasion and migration ability of C4-2 cells were detected by transwell assay in vitro and by small animal in vivo imaging experiments in vivo, the results showed that downregulate Ctbp2 expression could significantly recede the invasion and migration ability of C4-2 cells in vitro and in vivo. The m RNA and protein expression levels of E-cadherin were significantly elevated and the protein expression levels of N-cadherin was signigicantly decreased post Ctbp2 knocked out in C4-2 cells. CO-IP assay proved that Ctbp2 and slμg interactd with each other in C4-2 cells. Phalloidine staining showed that the constituent parts of cytoskeletal structure of C4-2 cells significantly changed post Ct BP2 downregulation. The expression levels of Rho C m RNA and protein, and the expression levels of ROCK1, P-MLC were significantly reduced post Ct BP2 downregulation in C4-2 cells. Conclusions:1. Ctbp2 was highly expressed in human prostate cancer tissues. 2. Downregulate the expression levels of Ct BP2 could inhibit the proliferation rate of C4-2 cells in vitro and in vivo, and promote C4-2 cells apoptosis. 3. Ct BP2 regulates the invasion and migration ability of C4-2 cells by EMT pasway and Rho C- ROCK1 pasway.