CtBP2对前列腺癌细胞生物学行为的影响

发布时间：2018-06-11 21:05

  本文选题：前列腺癌 + 羧基末端结合蛋白2　； 参考：《天津医科大学》2016年博士论文

【摘要】：目的:本研究通过shRNA干扰技术建立稳定低表达CtBP2的前列癌C4-2细胞,利用体内外实验技术检测CtBP2低表达对C4-2细胞增殖、凋亡及迁移侵袭能力的影响;并揭示CtBP2调控C4-2细胞上述生物学行为的具体机制。为未来临床靶向CtBP2基因治疗前列腺癌提供理论基础。方法:1.通过实时荧光定量聚合酶链反应(RT-qPCR)检测前列腺癌组织和癌旁组织中Ctbp2 mRNA表达量;通过免疫组化检测前列腺癌组织和癌旁组织中Ctbp2蛋白的表达水平;将靶向Ctbp2基因shRNA质粒及阴性对照(Vector)质粒分别转入C4-2细胞,RT-qPCR和western blot法分别检测Ctbp2 mRNA及蛋白的表达水平以检验治疗转染的效率;细胞计数试剂盒(CCK-8)法和流式细胞仪分别检测下调Ctbp2表达对C4-2细胞体外增殖能力和凋亡的影响;裸鼠皮下移植瘤实验观察下调Ctbp2对C4-2细胞体内成瘤能力的影响;利用Ctbp2特异性底物MTOB暴露C4-2细胞检测其对C4-2细胞增殖能力的影响,以此进一步验证Ctbp2表达对C4-2细胞体外增殖能力的影响;通过western blot实验检测下调Ctbp2表达水平对凋亡相关蛋白BIK表达水平的影响。2、通过Transwell实验检测下调Ctbp2对C4-2细胞体外侵袭(预铺胶)和迁移能力的影响;通过建立裸鼠皮下移植瘤,在细胞移植后第45天时利用小动物活体成像仪检测肿瘤细胞在胸腹腔脏器和骨骼等部位有无转移灶;通过RT-qPCR检测下调Ctbp2对C4-2细胞上皮标志分子E-cadherin mRNA表达水平的影响;通过western blot实验检测下调Ctbp2对C4-2细胞上皮标志分子E-cadherin和间质标志分子N-cadherin蛋白表达水平的影响;通过鬼笔环肽染色细胞骨架检测下调Ctbp2对C4-2细胞细胞骨架结构的影响;通过分析UCSC数据库中的信息,搜寻关于Ctbp2与RhoC之间存在相互作用关系的证据;通过RT-qPCR检测下调Ctbp2对C4-2细胞上皮标志分子RhoC mRNA表达水平的影响;通过western blot实验检测下调Ctbp2对C4-2细胞中RhoC-ROCK1-MLC信号通路中蛋白表达水平的影响。结果:1.Ctbp2 mRNA在前列腺癌组织中的表达水平显著高于癌旁正常组织(2.79±1.04比1.0±0.41)(p0.05);ctbp2蛋白质表达水平在前列腺癌组织中也较癌旁组织中显著增高;通过ctbp2-shrna转染c4-2细胞可显著降低细胞中ctbp2mrna和蛋白的表达水平;通过cck-8试剂盒检测稳定低表达ctbp2基因的c4-2细胞24小时内的增殖能力,发现降低ctbp2的表达水平可显著减弱c4-2细胞的增殖能力(0.59±0.11)(p0.05),而vector组细胞(0.58±0.09)的增殖能力与cont组c4-2细胞(0.34±0.05)间无显著差异,(p0.05);细胞克隆形成实验发现降低ctbp2的表达水平可显著减弱c4-2细胞的克隆形成能力;通过不同浓度mtob暴露c4-2细胞检测其对c4-2细胞增殖能力的影响,结果显示c4-2细胞增殖能力随着mtob浓度(0.1mm、0.5mm、1mm、4mm、7mm、10mm)的增加而逐渐降低(0.102±0.013,0.104±0.014,0.097±0.006,0.086±0.004,0.063±0.007,0.034±0.004,0.023±0.004);通过裸鼠皮下移植瘤实验发现,降低ctbp2的表达水平可显著减弱c4-2细胞在小鼠体内的增殖能力,细胞皮下移植后第35天ctbp2组和vector组小鼠的肿瘤体积分别为893.28±241.15mm3vs2173.83±472.71mm3(p0.05);通过annexinv-pi染色,利用流式细胞仪检测细胞的凋亡情况,结果发现下调ctbp2的表达水平可显著促进c4-2细胞的凋亡cont组ctbp2-shrna组凋亡细胞数量分别为10.10%±0.96%vs41.00%±3.01%(p0.05),而vector组和cont组之间无显著差异,(p0.05);通过westernblot实验发现降低ctbp2的表达水平可显著促进c4-2细胞中凋亡相关蛋白bik的表达水平。2.transwell实验检测降低ctbp2的表达水平对c4-2细胞的侵袭(预铺胶)和迁移能力发现,与cont组细胞相比较ctbp2-shrna组细胞的侵袭(63.25±6.75vs249.00±20.50)和迁移(805.50±63.25vs1639.25±71.38)能力均显著被抑制(p0.05),而vector组和cont组间无显著差异(p0.05);通过建立了皮下移植瘤裸鼠模型,并在细胞皮下移植后第45天时用小动物活体成像仪检测荷瘤小鼠体内转移灶的情况发现在10只vector组小鼠模型中检测到有3只鼠发生了不同程度的腹部脏器转移(主要为胃肠道系统),而在ctbp2-shrna组小鼠中未发现有腹部脏器转移灶;另外在vector组有4只小鼠发生了骨转移,转移部位主要集中在四肢,而ctbp2-shrna组小鼠中未发现有骨转移灶情况的发生。通过rt-qpcr和westernblot实验发现降低c4-2细胞中ctbp2的表达水平可显著增强上皮细胞分子标志物e-cadherinmrna和蛋白的表达水平,同时n-cadherin蛋白表达量下降;co-ip实验证实在c4-2细胞中ctbp2与e-cadherin转录因子slμg之间存在相互结合作用关系;通过鬼笔环肽染色细胞骨架发现,降低C4-2细胞中CtBP2的表达水平可显著减少细胞骨架成份;检索UCSC数据库得知CtBP2可结合在Rho C的启动子序列上,发挥转录因子的作用;通过RT-qPCR发现降低CtBP2的表达水平可使C4-2细胞中RhoC mRNA的表达量显著下降,同时通过western blot实验发现RhoC下游信号分子ROCK1和p-MLC的蛋白表达量也下降。结论:1.辅助转录因子CtBP2在前列腺癌组织中高表达;2.降低CtBP2的表达水平可显著抑制前列腺癌C4-2细胞的体内外增殖能力并促进其凋亡;3.降低CtBP2的表达水平可显著抑制前列腺癌C4-2细胞的体内外侵袭迁移能力,且在体外CtBP2可通过EMT通路和RhoC-ROCK1通路调控C4-2细胞的侵袭迁移能力。
[Abstract]:Objective: to establish a prostex cancer C4-2 cell with low expression of CtBP2 by shRNA interference technique, and to detect the effect of CtBP2 low expression on the proliferation, apoptosis and migration and invasion of C4-2 cells in vivo and in vitro, and to reveal the specific mechanism of CtBP2 regulating the biological behavior of C4-2 cells in the future, and for the future clinical targeting of CtBP2 gene therapy. The theoretical basis of prostate cancer is provided. Methods: 1. the expression of Ctbp2 mRNA in prostate cancer tissue and para cancer tissue was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and the expression of Ctbp2 protein in prostate cancer tissue and para cancer tissue was detected by immunohistochemistry; the target Ctbp2 gene shRNA plasmid and negative control (Vector) substance were targeted. The expression level of Ctbp2 mRNA and protein was detected by RT-qPCR and Western blot, respectively, to test the efficiency of transfection, respectively. Cell count Kit (CCK-8) and flow cytometry were used to detect the effect of down regulated Ctbp2 expression on the proliferation and apoptosis of C4-2 cells in vitro, and to observe the down-regulation of Ctbp in nude mice by subcutaneous transplantation of nude mice. 2 Effect on the tumor formation ability of C4-2 cells in vivo; the effect of C4-2 cells on the proliferation ability of C4-2 cells by Ctbp2 specific substrate MTOB was detected to further verify the effect of Ctbp2 expression on the proliferation ability of C4-2 cells in vitro; the BIK expression level of down regulated Ctbp2 tables was detected by Western blot test. The effect of.2 was detected by Transwell test. The effect of down-regulation of Ctbp2 on the invasion (pre paving) and migration ability of C4-2 cells in vitro was detected. By establishing subcutaneous transplanted tumor in nude mice, the tumor cells were detected by the small animal living imager at forty-fifth days after the transplantation, and the tumor cells were detected in the thoracic and abdominal organs and the bone and other parts of the bone, and the Ctb was down regulated by RT-qPCR. The effect of P2 on the expression level of C4-2 cell epithelial marker molecule E-cadherin mRNA; the effect of down regulated Ctbp2 on the expression of E-cadherin and interstitial marker molecule N-cadherin protein expression level of C4-2 cell epithelial marker molecules by Western blot test; the cytoskeleton structure of C4-2 cells was detected by cytoskeleton through the cytoskeleton of phallus cytosin staining. Influence; through the analysis of the information in the UCSC database, search for evidence about the interaction between Ctbp2 and RhoC, and the effect of Ctbp2 on the expression level of RhoC mRNA of the C4-2 cell epithelial markers by RT-qPCR detection; and the expression of protein expression in the signaling pathway in the C4-2 cells by Western blot experiment Results: the expression level of 1.Ctbp2 mRNA in prostate cancer tissues was significantly higher than that of normal tissue adjacent to the carcinoma (2.79 + 1.04 to 1 + 0.41) (P0.05), and the expression level of ctbp2 protein was significantly higher in the prostate cancer tissue than in the para cancerous tissue, and the ctbp2mrna and protein in the cells could be significantly reduced by ctbp2-shrna transfer to C4-2 cells. The proliferation ability of C4-2 cells with stable low expression of ctbp2 gene in 24 hours was detected by CCK-8 kit, and it was found that the expression level of ctbp2 decreased significantly (0.59 + 0.11) (P0.05), while there was no significant difference between the proliferation ability of vector group (0.58 + 0.09) and C4-2 cells (0.34 + 0.05) in cont group (P0.05). The cell clone formation experiment found that reducing the expression level of ctbp2 could significantly weaken the cloning and formation ability of C4-2 cells, and the effect on the proliferation ability of C4-2 cells was detected by C4-2 cells exposed to different concentrations of mtob. The results showed that the proliferation ability of C4-2 cells gradually decreased with the increase of mtob concentration (0.1mm, 0.5mm, 1mm, 4mm, 7mm, and 7mm). 0.013,0.104 + 0.014,0.097 + 0.006,0.086 + 0.004,0.063 + 0.007,0.034 + 0.004,0.023 + 0.004). By subcutaneous transplantation of nude mice, it was found that reducing the expression level of ctbp2 could significantly reduce the proliferation ability of C4-2 cells in mice, and the volume of tumor in ctbp2 group and vector group was 893.28 + 241.1 after thirty-fifth days after subcutaneous transplantation. 5mm3vs2173.83 + 472.71mm3 (P0.05); the apoptotic cells were detected by flow cytometry with annexinv-pi staining. The results showed that the number of apoptotic cells in the cont group of C4-2 cells was 10.10% + 0.96%vs41.00% + 3.01% (P0.05), respectively, and there was no significant difference between the vector group and the cont group. The difference, (P0.05); through the Westernblot experiment, it was found that reducing the expression level of ctbp2 significantly promoted the expression level of apoptosis related protein bik in C4-2 cells,.2.transwell test reduced the invasion (pre paving) and migration of C4-2 cells by ctbp2 expression level, and compared the invasion of the ctbp2-shrna group with the cont group cells (63.25 The ability of + 6.75vs249.00 + 20.50) and migration (805.50 + 63.25vs1639.25 + 71.38) were significantly inhibited (P0.05), but there was no significant difference between group vector and cont group (P0.05). A nude mouse model of subcutaneous transplantation tumor was established and the metastasis of tumor bearing mice was detected by small animal living imaging instrument at forty-fifth days after subcutaneous transplantation. In 10 vector mice, 3 rats were detected in different degrees of abdominal organ transfer (mainly the gastrointestinal tract), but no abdominal organ metastasis was found in group ctbp2-shrna mice; and 4 mice in group vector had bone metastases, and the metastatic sites were mainly concentrated on the limbs, while in group ctbp2-shrna mice were not found. RT-qPCR and Westernblot experiments showed that the expression level of ctbp2 in C4-2 cells decreased significantly, and the expression level of e-cadherinmrna and protein in the epithelial cell markers was significantly enhanced, while the expression of N-cadherin protein was decreased, and co-Ip experiments confirmed ctbp2 and E-cadherin transcription factor SL g in C4-2 cells. There is a binding relationship between the C4-2 cells and the cytoskeleton through the cytoskeleton. The reduction of the CtBP2 expression level in the C4-2 cells can significantly reduce the cytoskeleton component; the retrieval of the UCSC database shows that CtBP2 can be used in the promoter sequence of Rho C, and the expression level of CtBP2 can be reduced by RT-qPCR. The expression of RhoC mRNA in C4-2 cells decreased significantly, and the protein expression of ROCK1 and p-MLC in the downstream signal molecules of RhoC decreased by Western blot. Conclusion: the 1. auxiliary transcription factor CtBP2 is highly expressed in the prostate cancer tissue, and 2. to reduce the expression level of CtBP2 can significantly inhibit the proliferation of prostate cancer C4-2 cells in vivo and in vitro 3. reduced CtBP2 expression level significantly inhibited the invasion and migration of C4-2 cells in vitro and in vitro, and in vitro CtBP2 could regulate the invasion and migration of C4-2 cells through the EMT pathway and the RhoC-ROCK1 pathway.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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