【作者】 刘兆宇；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 生化与分子生物学， 2010， 硕士

【摘要】 Rhox5是Rhox同源异型框基因簇中的主要成员,其在胚胎发育、生殖系统的发育、精子的形成和成熟过程中发挥着重要作用。有研究表明,Rhox5在多种肿瘤细胞中存在异常表达,但其具体功能和作用机制仍不清楚。实验室前期工作利用酵母双杂交技术发现Rhox5与前列腺癌相关蛋白PSAP存在相互作用。这提示,Rhox5可能在前列腺癌的发展进程中扮演着某种角色。基于此,本课题首次将Rhox5与前列腺癌关联,研究Rhox5对前列腺癌细胞增殖和凋亡的影响,并初步探讨其可能的作用机制。首先,我们采用RT-PCR和Western Blot的方法确认Rhox5在前列腺癌细胞TRAMP-C2中的表达。然后,通过荧光筛选和Western Blot筛选得到靶向Rhox5的有效siRNA序列,并以此为基础构建Rhox5的shRNA真核表达载体,建立Rhox5稳定沉默前列腺癌细胞系。然后,我们采用MTT法检测稳定细胞系的增殖以及P13K特异性抑制剂LY294002预处理后细胞的增殖情况；用Annexin V/PI双染法流式细胞仪检测无血清诱导条件下的细胞凋亡情况；并用Western Blot检测稳定细胞系中Akt的磷酸化水平。结果发现,与对照组相比,稳定沉默Rhox5的前列腺癌细胞增殖明显加快(p<0.01),而LY294002预处理后则较对照组无明显差异；此外,稳定沉默Rhox5的前列腺癌细胞的凋亡率明显降低(p<0.05),Akt磷酸化水平明显升高。这些结果表明,Rhox5可以抑制前列腺癌细胞增殖并促进其凋亡,而这可能是通过PI3K/Akt信号通路进行调控的。为进一步探讨Rhox5功能发挥的作用机制,我们用Co-IP的方法研究了Rhox5与PSAP在前列腺癌细胞中的内源性相互作用,并用Western Blot检测了稳定前列腺癌细胞系培养上清中PSAP的表达以及PSAP对PI3K/Akt信号通路的活化作用。结果发现,Rhox5在前列腺癌细胞中可以与PSAP内源性相互作用；Rhox5的沉默上调了培养上清中PSAP的表达；此外,PSAP还被证实可以在鼠前列腺癌细胞中激活PI3K/Akt信号通路。这些结果提示,Rhox5可能通过与PSAP结合抑制PSAP的胞外分泌,从而抑制由PSAP诱导的PI3K/Akt信号通路的活化,进而抑制前列腺癌细胞的增殖、促进其凋亡。但这尚需进一步实验验证。综上所述,本研究首次发现了Rhox5在前列腺癌细胞中的表达,并发现Rhox5能够抑制前列腺癌细胞增殖并促进其凋亡,而这可能是通过PI3K/Akt信号通路进行调控的。此外,本研究还发现Rhox5在前列腺癌细胞中可以与PSAP内源性相互作用,且Rhox5影响PSAP在细胞培养上清中的表达,从而提出Rhox5可能通过与PSAP结合抑制PSAP的胞外分泌而实现对前列腺癌细胞的增殖抑制和凋亡促进作用。更多还原

【Abstract】 Rhox5 is the founding member of Rhox homeobox gene cluster, and it plays important roles in embryonic development, reproductive development and spermatogenesis. Although studies have indicated that Rhox5 is aberrantly expressed in multiple tumor cells, its function and mechanism are still unclear. Our lab previously found the interaction between Rhox5 and PSAP, a prostate cancer related protein, by yeast-two hybrid method, which suggests that Rhox5 may play a role in prostate cancer development. Hence, for the first time we associate Rhox5 with prostate cancer, to study its influence on proliferation and apoptosis of prostate cancer cells and primitively explore its potential mechanism.We first identified the expression of Rhox5 in prostate cancer cell line TRAMP-C2 by RT-PCR and Western Blot. Then, we obtained effective siRNA against Rhox5 by fluorescence screening and Western Blot screening, based on which we then constructed a eukaryotic shRNA expression vector of Rhox5 and established a stable Rhox5-silencing prostate cancer cell line. The proliferation of stable cell lines with/without LY294002 pretreatment was detected by MTT assay; apoptosis induced by serum starvation was detected by Annexin V/PI double staining-FCM method; phosphorylation level of Akt was dectected by Western Blot analysis. Our results show that the proliferation of Rhox5-silencing prostate cancer cells was significantly increased as compared to control (p<0.01), while LY294002 pretreatment abrogated this difference; in addition, the apoptosis rate of Rhox5-silencing prostate cancer cells was obviously reduced (p<0.01), and the phosphorylation level of Akt was increased. These results suggest that Rhox5 can inhibit the proliferation of prostate cancer cells and promote their apoptosis, which might be mediated via PI3K/Akt pathway.In order to explore the mechanism that Rhox5 functions, we studied the endogenous interaction between Rhox5 and PSAP in prostate cancer cells by coimmunoprecipitation, and we detected the expression of PSAP in the culture media of stable prostate cancer cell lines and confirmed the activity of PSAP on PI3K/Akt pathway. Our results show that Rhox5 can endogenously interact with PSAP in prostate cancer cells and Rhox5 silencing upregulates the expression of PSAP in the culture media. Besides, we have confirmed that PSAP can activate PI3K/Akt pathway in mouse prostate cancer cells. All these results together suggest that Rhox5 may inhibit the secretion of PSAP by binding to it, which inhibits the PSAP-induced activation of PI3K/Akt pathway and further inhibits the proliferation and promotes apoptosis of prostate cancer cells. However, further experiments are needed to confirm this hypothesis.In conclusion, our study for the first time has found the expression of Rhox5 in prostate cancer cells and the ability of Rhox5 to inhibit the proliferation and promote apoptosis of prostate cancer cells, which might be regulated via PI3K/Akt pathway. In addition, we have found that Rhox5 can endogenously interact with PSAP in prostate cancer cells and that Rhox5 affects the expression of PSAP in the culture medium. Therefore, we propose that Rhox5 may inhibit proliferation and promote apoptosis of prostate cancer cells by binding to PSAP and inhibiting its secretion.更多还原