【作者】 李文杰；

【导师】 桂耀庭；

【作者基本信息】 汕头大学 ， 生殖生理学， 2011， 硕士

【摘要】 目的：探讨原钙粘附蛋白10（Procadherin 10, PCDH10）基因在前列腺癌细胞系中的表达特征与调控机制。方法：采用半定量RT-PCR、荧光定量RT-PCR、蛋白印迹（Western Blotting）、免疫荧光染色法、甲基化特异性PCR（MSP）或亚硫酸盐基因组测序（BGS）等方法，检测PCDH10在前列腺癌组织或四种细胞系（22RV1、PC3、DU145和LNCaP）中的表达特征及启动子甲基化的状态。采用DNA甲基转移酶抑制剂5-杂氮脱氧胞嘧啶核苷（AZA）和组蛋白去乙酰化酶抑制剂曲古霉素A（TSA）处理前列腺癌细胞系，以探讨DNA甲基化和组蛋白去乙酰化对PCDH10基因转录的调控作用。结果：PCDH10 mRNA在人子宫、卵巢、甲状腺、食道、胃、肝、前列腺、睾丸和肾组织中均有表达，在前列腺组织中表达较高。与正常前列腺组织相比，PCDH10在前列腺癌细胞系中的表达明显减少或消失，其表达量与PCDH10启动子甲基化呈负相关。四种前列腺癌细胞系经AZA处理后，PCDH10在22RV1、PC3和LNCaP细胞系中启动子甲基化的密度均显著降低，其mRNA的表达明显升高；TSA处理后，PCDH10 mRNA在LNCaP细胞系的表达明显增加，而在22RV1、PC3和DU145三种细胞系中的表达无显著差异；将AZA与TSA联合使用，结果显示，两者对PCDH10 mRNA的表达具有协同作用。结论：PCDH10在前列腺癌细胞系中的表达明显减少或消失，其下降程度与PCDH10启动子CpG岛的甲基化状态呈负相关。PCDH10在前列腺癌细胞系中启动子甲基化和组蛋白去乙酰化可能是PCDH10低表达的分子机理。更多还原

【Abstract】 Objectives：To investigate the expression and regulation of PCDH10 in prostate cancer celllines.Methods: The expression and promoter methylation status of PCDH10 in four prostate cancercell lines (22RV1、PC3、DU145 and LNCaP) were analyzed by RT-PCR, real-time RT-PCR,Western Blotting, Immunofluorescence staining, methylation-specific PCR and bisulfatesequencing. The roles for DNA methylation and histone deacetylation on the expression ofPCDH10 were studied by the treatment of DNA methyltransferase inhibitor 5-azacytidine (AZA)and histone deacetylase inhibitor Trichostatin A (TSA) in prostate cancer cell lines.Results：PCDH10 mRNA was widely expressed in human tissues with a higher level in prostatetissue. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell linescompared with normal prostate tissue. PCDH10 expression was negatively correlated with themethylation status of PCDH10 promoter. Furthermore, after AZA treatment, PCDH10 promotermethylation was significantly decreased while mRNA level was obviously elevated in threeprostate cancer cell lines (22RV1、PC3 and LNCaP); However, the expression of PCDH10mRNA was enhanced only in LNCap cell line while there was no significant differences amongthe other cell lines after TSA treatment; When combined AZA and TSA, the overall effect of thiscomplex on PCDH10 mRNA was synergetic.Conclusion: The expression of PCDH10 was decreased in prostate cancer cell lines, which wasnegatively associated with the aberrant methylation in PCDH10 promoter. DNA methylation andhistone deacetylase were two crucial molecular events leading to lower expression of PCDH10in prostate cancer cell lines.更多还原