CtBP2-ROCK通路调节前列腺癌细胞侵袭

发布时间：2018-05-31 12:06

本文选题：前列腺癌 + CTBP2　；参考：《天津医科大学》2014年硕士论文

【摘要】：目的明确CTBP2表达水平及位置改变对前列腺癌细胞体外生物学行为的影响,并初步探讨其分子机制。方法运用免疫组织化学检测CTBP2在前列腺增生与前列腺癌组织中的表达水平及位置改变；分别用westernblot与RT-qPCR检测CTBP2在正常前列腺细胞RWPE-1与前列腺癌细胞PC3中的蛋白水平与mRNA表达量,利用免疫荧光及激光共聚焦显微镜检测CTBP2在这些细胞中的位置改变;构建CTBP2表达质粒,并用其转化感受态细胞,经扩增后提取高浓度质粒,随后将其转染入PC3细胞中,经G418筛选及单克隆细胞培养后获得稳定转染株；分别应用RT-qPCR及Westernblot验证转染后细胞中CtBP2的mRNA表达量以及蛋白水平；使用MTT法检测CTBP2高表达前列腺癌细胞增殖能力变化,Transwell法检测CTBP2高、低表达前列腺癌细胞侵袭能力变化,磷脂酰丝氨酸外翻法分析(Annexin V)检测CTBP2高、低表达前列腺癌细胞凋亡改变;利用RT-qPCR检测可能受CTBP2调节的侵袭相关分子mRNA表达量,进一步运用westernblot验证RT-qRCR所得结果及该效应分子下游靶蛋白的改变。整理数据,所有需要统计分析的实验结果均运用SPSS软件进行统计分析。结果前列腺癌组织中CTBP2蛋白水平增加,且与肿瘤分期、gleason评分成正相关,CTBP2在前列腺增生组织及局限性前列腺癌组织中主要位于细胞核中,而在转移性前列腺癌组织胞浆中也出现；与正常前列腺细胞RWPE-1相比,前列腺癌细胞PC3中CTBP2mRNA表达量与蛋白水平增加,在RWPE-1中CTBP2主要位于细胞核中,而其在PC3细胞核与细胞浆中均表达；取CTBP2表达质粒转化感受态细胞,经扩增后成功提取高浓度质粒；通过脂质体介导将CTBP2表达质粒转染入PC3细胞,经G418筛选与单克隆细胞培养获得CTBP2稳定表达细胞株,并验证该细胞株CTBP2稳定高表达；细胞生物学行为检测表明CTBP2高表达可显著提高前列腺癌细胞侵袭能力,但对细胞增殖与凋亡无显著影响；而CTBP2低表达可显著降低细胞侵袭能力,增加细胞凋亡。RT-qPCR发现CTBP2低表达可降低前列腺癌细胞中ROCK1表达,westernblot检测也发现CTBP2低表达细胞中ROCK1蛋白水平降低,这与RT-qRCR结果一致。Westernbolt检测发现ROCK1下游信号分子p-MLC与c-Myc蛋白水平也降低,且c-Myc下游分子HSPC111蛋白含量降低。结论 CTBP2表达水平及位置改变可影响前列腺癌细胞体外生物学行为,前列腺癌细胞中可能存在CTBP2-ROCK通路,其可能是促进前列腺癌细胞侵袭的机制之一。
[Abstract]:Purpose To investigate the effect of CTBP2 expression level and location on the biological behavior of prostate cancer cells in vitro, and to explore its molecular mechanism. Method Immunohistochemistry was used to detect the expression and location of CTBP2 in prostatic hyperplasia and prostate cancer, and westernblot and RT-qPCR were used to detect the protein level and mRNA expression of CTBP2 in RWPE-1 and PC3, respectively. The position of CTBP2 in these cells was detected by immunofluorescence and laser confocal microscopy, the expression plasmid of CTBP2 was constructed and transformed into the competent cells, the high concentration plasmid was extracted after amplification, and then transfected into PC3 cells. The stable transfection strain was obtained by G418 screening and monoclonal cell culture, and the mRNA expression and protein level of CtBP2 in transfected cells were verified by RT-qPCR and Westernblot, respectively. The proliferative ability of prostate cancer cells with high expression of CTBP2 was detected by MTT assay. The invasion ability of prostate cancer cells with low expression of CTBP2 was detected by Transwell method. The changes of apoptosis of prostate cancer cells with low expression were detected by Annexin V and high CTBP2 by Phosphatidyl serine valgus assay. RT-qPCR was used to detect the mRNA expression of invasion-related molecules which may be regulated by CTBP2. Westernblot was used to verify the results of RT-qRCR and the changes of downstream target proteins of the effector molecule. All the experimental results that need statistical analysis are analyzed by SPSS software. Result The level of CTBP2 protein was increased in prostate cancer tissues and positively correlated with tumor staging. CTBP2 was mainly located in the nucleus of prostate hyperplasia and localized prostate cancer tissues, but also in the cytoplasm of metastatic prostate cancer. Compared with the normal prostatic cell RWPE-1, the expression of CTBP2mRNA and protein in PC3 of prostate cancer cell increased. In RWPE-1, CTBP2 was mainly expressed in the nucleus, but it was expressed in the nucleus and cytoplasm of PC3. After amplification, the high concentration plasmid was extracted and the CTBP2 expression plasmid was transfected into PC3 cells mediated by liposome. The stable expression of CTBP2 was obtained by G418 screening and monoclonal cell culture, and the stable expression of CTBP2 in the cell line was verified. The results of cell biological behavior showed that the high expression of CTBP2 could significantly increase the invasive ability of prostate cancer cells, but had no significant effect on cell proliferation and apoptosis, while the low expression of CTBP2 could significantly reduce the invasive ability of prostate cancer cells. Increasing apoptosis. RT-qPCR found that low expression of CTBP2 could decrease ROCK1 expression in prostate cancer cells. Western blot analysis also found that ROCK1 protein level in CTBP2 low expression cells decreased, which was consistent with the results of RT-qRCR. Western bolt detection showed that the lower ROCK1 signal molecules p-MLC and c-Myc protein levels were also decreased. The content of HSPC111 protein in the downstream of c-Myc was decreased. Conclusion The change of CTBP2 expression level and location may affect the biological behavior of prostate cancer cells in vitro. There may be a CTBP2-ROCK pathway in prostate cancer cells, which may be one of the mechanisms to promote the invasion of prostate cancer cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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