EpCAM与雄激素非依赖性前列腺癌发生的相关性研究

发布时间：2018-07-09 17:32

本文选题：上皮细胞粘附分子 + 雄激素抵抗性前列腺癌　；参考：《第四军医大学》2014年博士论文

【摘要】：雄激素抵抗性前列腺癌是前列腺癌致死的主要原因。关于雄激素抵抗性前列腺癌发病机制的研究目前已经成为前列腺癌研究的热点之一。上皮细胞黏附分子(Epithelial cell adhesion molecule，EpCAM)是一类I型跨膜糖蛋白，其在正常组织中只局限性的表达在上皮组织的基底膜处，但在快速增殖的肿瘤细胞中则大量表达。EpCAM在肿瘤组织中的过度表达与肿瘤的发生、发展及预后密切相关，已经成为肿瘤诊断和靶向治疗重要的候选分子。关于EpCAM在前列腺癌组织中的表达与雄激素抵抗性前列腺癌的发生相关性的研究目前还鲜有报道。本研究利用免疫组织化学和前列腺癌组织芯片技术在6例正常前列腺组织、24例前列腺增生组织及210例前列腺癌组织中检测了EpCAM蛋白的表达差异。结果显示：EpCAM蛋白在正常前列腺组织表达量较低且主要局限性表达在前列腺管腔上皮细胞的基底膜处，在管腔上皮的细胞间连接处仅有极少量表达。在前列腺增生组织中，EpCAM的表达量增高，其表达不仅在管腔上皮组织的基底膜处且在管腔上皮细胞间连接处表达量也较高。在前列腺癌组织中，EpCAM的表达量显著升高，且弥散的表达在前列腺管腔上皮细胞的各个部分，除了上皮细胞的基底部和细胞间连接处有大量的表达以外，在管腔上皮细胞的顶端也有大量的表达。Western-blot在前列腺癌组织及相应的癌旁组织中检测EpCAM的表达,结果进一步肯定了其在前列腺癌组织中的表达量显著高于癌旁组织。统计学分析结果显示：EpCAM蛋白的高表达与前列腺癌的临床病理分级、淋巴结转移、远处转移以及手术前血清中总的前列腺特异性抗原（Prostatespecific antigen，PSA）水平等具有明显相关性，且EpCAM高表达的前列腺癌患者，其无转移生存期和总生存期均显著短于EpCAM低表达的前列腺癌患者。 Western-blot检测四种前列腺癌细胞系DU-145，PC-3，LNCaP及VCaP中EpCAM的表达，结果显示：LNCaP细胞中EpCAM的表达量最高，其次为PC-3细胞、DU-145细胞和VCaP细胞。采用shRNA干扰技术即利用EpCAM-shRNA慢病毒颗粒及相应的慢病毒shRNA载体颗粒感染前列腺癌LNCaP细胞并建立稳定转染的细胞系EpCAM-shRNA5和对照细胞系Vector3。在EpCAM-shRNA5和Vector3细胞中分别检测雄激素受体（Androgen receptor，AR）及PSA的表达水平，结果显示：EpCAM-shRNA5细胞中，AR和PSA的表达水平明显低于Vector3细胞。ELISA法检测EpCAM-shRNA5细胞培养上清中的PSA水平同样低于Vector3细胞。MTT法、TUNEL法、Annexin V/PI双染法、细胞划痕实验以及Transwell实验分别用来在体外检测EpCAM-shRNA5和Vector3细胞的增殖、抗凋亡、迁移以及侵袭能力，结果显示EpCAM-shRNA5细胞的增殖、抗凋亡、迁移以及侵袭能力均显著低于对照组Vector3细胞。采用小鼠体内肿瘤种植模型进一步在体内检测两种细胞的肿瘤形成能力，结果显示EpCAM-shRNA5细胞的体内成瘤能力明显低于Vector3细胞。我们的研究结果提示：EpCAM在前列腺癌组织中的过度表达可能通过促进前列腺癌细胞中AR的表达来使得前列腺癌细胞能够适应去势雄激素水平而继续存活。对EpCAM-shRNA5和Vector3细胞进行体外培养时可以观察到Vector3细胞与LNCaP细胞相比形态变化不大，即均为纺锤状，并有细丝状的伪足，细胞间连接几乎不存在。而EpCAM-shRNA5细胞形态则由细长的纺锤状变为较规则的卵石状，细丝状伪足消失，且细胞间出现了比较稀疏的细胞间连接。这些形态变化提示EpCAM的表达可能促进上皮-间质转化（Epithelial-mesenchymal transition，，EMT）。进一步在EpCAM-shRNA5和Vector3细胞中检测EMT发生相关的蛋白标志物,结果显示：与Vector3细胞相比EpCAM-shRNA5细胞中上皮细胞特异性蛋白E-cadherin和Cytokeratin的表达增高而间质细胞特异性蛋白Vimentin和Fibronectin表达降低。利用免疫荧光染色法进一步肯定了EpCAM可促进EMT的发生。由于EMT的发生会赋予细胞干细胞的特性，同时EpCAM在一些肿瘤组织中也被看作干细胞的标记物。因此，我们进一步验证EpCAM-shRNA5和Vector3细胞是否具有干细胞方面的特性。首先，利用平板克隆形成实验检测两种细胞的克隆形成能力,结果显示：EpCAM-shRNA5细胞的克隆形成能力显著低于Vector3。进一步检测干细胞相关的转录因子OCT4, SOX2, NANOG的表达,结果显示：EpCAM-shRNA5细胞中三种干细胞相关转录因子的表达水平明显低于Vector3细胞。提示EpCAM不仅可以通过上调AR水平来提高前列腺癌细胞对去势水平雄激素的耐受，同时还可以通过促进EMT的发生及肿瘤细胞的干细胞特性的表达来提高前列腺癌细胞在去势水雄激素平下的生存能力。综上所述，我们的研究首次将EpCAM在前列腺组织中的过表达与雄激素抵抗性前列腺癌的发生相联系起来。证实EpCAM可通过促进AR的表达，EMT的发生及干细胞特性的形成等来促进前列腺癌细胞在去势雄激素水平下的生存能力，从而促进雄激素抵抗性前列腺癌的发生。我们的实验数据为雄激素抵抗性前列腺癌的免疫治疗提供了重要的候选靶分子。
[Abstract]:Androgen resistance to prostate cancer is the main cause of the death of prostate cancer. Research on the mechanism of androgen resistance to prostate cancer is now one of the hotspots in the research of prostate cancer. Epithelial cell adhesion molecule (EpCAM) is a class of I type transmembrane glycoprotein, which is only in normal tissues. Limited expression is located at the basal membrane of epithelial tissue, but the excessive expression of.EpCAM in the tumor cells in the rapidly proliferating tumor cells is closely related to the occurrence, development and prognosis of tumor. It has become an important candidate for the diagnosis of tumor and target therapy. The expression of EpCAM in the prostate cancer tissue and the male excitation There is little report on the correlation between prostate cancer and prostate cancer.
In this study, the expression of EpCAM protein was detected in 6 normal prostate tissues, 24 cases of benign prostatic hyperplasia and 210 cases of prostate cancer. The results showed that the expression of EpCAM protein in the normal prostate tissue was low and the main limitation was expressed in the prostatic cavity. In the basal membrane of the skin cells, only a very small amount of expression was found in the intercellular junction of the lumen epithelium. In the prostatic hyperplasia tissue, the expression of EpCAM increased. The expression of EpCAM was not only in the basal membrane of the epithelial tissue of the lumen but also in the intercellular junction of the lumen, and the expression of the expression was significantly increased in the adenocarcinoma of the anterior gland. The expression of diffuse expression in all parts of the epithelial cells of the prostate gland, except for the large amount of expression in the basal part of the epithelial cells and the intercellular junction, also has a large number of expressions of.Western-blot at the top of the epithelial cells of the lumen. The expression of EpCAM in the prostate cancer tissue and the corresponding para cancerous tissue is detected. The result further affirms its expression. The expression in the prostate cancer tissue was significantly higher than that in the paracancerous tissue. The statistical analysis showed that the high expression of EpCAM protein was correlated with the clinicopathological classification, lymph node metastasis, distant metastasis and the level of Prostatespecific antigen, PSA in the serum before operation, and Ep CAM patients with high expression of prostate cancer had no metastasis survival and overall survival time significantly shorter than those with low EpCAM expression.
Western-blot was used to detect the expression of EpCAM in four kinds of prostate cancer cell lines, DU-145, PC-3, LNCaP and VCaP. The results showed that the expression of EpCAM was the highest in LNCaP cells, followed by PC-3, DU-145 and VCaP cells. The expression level of androgen receptor (Androgen receptor, AR) and PSA in EpCAM-shRNA5 and Vector3 cells was detected in the cancer LNCaP cells and the stable transfected cell line EpCAM-shRNA5 and the control cell line Vector3.. The results showed that the expression level of AR and PSA was significantly lower than that in the EpCAM-shRNA5 cells. The level of PSA in NA5 cell culture supernatant was also lower than that of Vector3 cell.MTT method. TUNEL, Annexin V/PI double staining, cell scratch test and Transwell experiment were used to detect the proliferation, anti apoptosis, migration and invasion ability of EpCAM-shRNA5 and Vector3 cells in vitro, and the fruit showed the proliferation, anti apoptosis and migration of EpCAM-shRNA5 cells. Vector3 cells were significantly lower than the control group. The tumor formation ability of two cells was further detected in vivo by the tumor planting model in mice. The results showed that the tumor formation ability of EpCAM-shRNA5 cells in vivo was significantly lower than that of Vector3 cells. Our results suggest that the overexpression of EpCAM in the prostate cancer tissue It is possible that prostate cancer cells can adapt to castrated androgen levels and continue to survive by promoting the expression of AR in prostate cancer cells.
In vitro culture of EpCAM-shRNA5 and Vector3 cells can be observed that Vector3 cells have little change in morphology compared with LNCaP cells, that is, spindle shaped, and filamentous pseudo feet, with almost no intercellular connections, and EpCAM-shRNA5 cells form a slender spindle shape to a more regular pebbles, and filamentous pseudo foot disappears. There are relatively sparse intercellular connections between cells. These morphological changes suggest that the expression of EpCAM may promote epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT). The protein markers associated with EMT in EpCAM-shRNA5 and Vector3 cells are further detected, and the results show that compared with Vector3 cells in EpCAM-shRNA5 cells. The expression of epithelial cell specific protein E-cadherin and Cytokeratin increased while the expression of interstitial cell specific protein Vimentin and Fibronectin decreased. Using immunofluorescence staining, it was further affirmed that EpCAM could promote the occurrence of EMT. As EMT occurs, the characteristics of cell stem cells are given, and EpCAM is also seen in some tumor tissues. As a marker of stem cells, we further verify whether EpCAM-shRNA5 and Vector3 cells have stem cell characteristics. First, the clone formation ability of two cells was detected by a flat clone formation test. The results showed that the clone formation ability of EpCAM-shRNA5 cells was significantly lower than that of Vector3. for further detection of stem cells. The expression of transcription factors, OCT4, SOX2, NANOG, showed that the expression level of three stem cell related transcription factors in EpCAM-shRNA5 cells was significantly lower than that of Vector3 cells. It suggested that EpCAM not only improve the tolerance of prostate cancer cells to the castrated androgens by up regulation of AR, but also by promoting the occurrence of EMT and the development of EMT. The expression of stem cell characteristics of cancer cells enhances the survival of prostate cancer cells in androgen.
To sum up, our study is the first to link the overexpression of EpCAM in the prostate tissue to the occurrence of androgen resistance to prostate cancer. It is confirmed that EpCAM promotes the viability of prostate cancer cells at the level of androgens by promoting the expression of AR, the occurrence of EMT and the formation of stem cell characteristics, and thus promotes male prostatic cancer. Our data provide an important candidate for immunotherapy of androgen resistant prostate cancer.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

【引证文献】