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[目的]研究子宫内膜癌中孕激素受体亚型PRA、PRB表达与其启动子区甲基化状态的关系,探讨子宫内膜癌组织、细胞分子变化特征及孕激素受体亚型表达下调的机制。[方法](1)应用免疫组化SP法检测子宫内膜癌组织中PRA、PRB蛋白的表达。(2)应用甲基化特异性PCR检测子宫内膜癌组织及经5-杂氮脱氧胞苷(5′-aza-2′-deoxyóytidine ADC)处理前后子宫内膜癌Hec-1-B细胞系中PRA、PRB的甲基化状态。(3)应用免疫印迹法检测ADC处理Hec-1-B细胞系前后PRA、PRB蛋白的表达。[结果](1)与正常子宫内膜组织比较:PRA蛋白在高、中低分化子宫内膜癌组织中表达差异无统计学意义;PRB蛋白在高分化子宫内膜癌组织中表达无明显降低,差异无统计学意义,在中低分化子宫内膜癌组织中表达明显降低,差异有统计学意义。(2)与正常子宫内膜组织比较,子宫内膜癌组织中,PRA启动子区CpG岛甲基化率较低,差异无统计学意义,PRB启动子区CpG岛甲基化率较高,差异有统计学意义,与组织分化程度有关,与病理类型及有无淋巴结转移和有无肌层浸润无关;子宫内膜癌Hec-1-B细胞系中,加入ADC前PRA出现非甲基化条带,PRB出现甲基化条带,加入ADC后,PRA非甲基化条带不变,PRB甲基化条带消失。(3)子宫内膜癌Hec-1-B细胞系中加入不同浓度ADC后,PRA蛋白表达无明显改变,PRB蛋白表达不同程度增加。[结论]子宫内膜癌组织以及Hec-1-B细胞系中,存在PR基因异常甲基化,与PR表达下调关系密切,编码PR基因启动子的选择性B亚型甲基化,导致了PRB的失活,继而PRB蛋白表达下降或消失,可能参与了子宫内膜癌的形成。ADC可通过去甲基化作用使PRB恢复表达,可能提高子宫内膜癌的临床内分泌治疗效果。
[Objective] To investigate the relationship between the expression of PRA and PRB and the methylation status of its promoter region in endometrial carcinoma and to explore the molecular characteristics and the downregulation of progesterone receptor subtypes in endometrial carcinoma Mechanisms. [Method] (1) Immunohistochemical SP method was used to detect the expression of PRA and PRB in endometrial carcinoma tissues. (2) Methylation-specific PCR was used to detect endometrial cancer tissue and endometrial carcinoma Hec-1-B cell line before and after treatment with 5-aza-2’-deoxyóytidine ADC PRA, PRB methylation status. (3) Western blotting was used to detect the expression of PRA and PRB protein before and after treatment of Hec-1-B cell line with ADC. [Results] (1) Compared with normal endometrium, the expression of PRA protein in high and moderately poorly differentiated endometrial carcinoma was not statistically different; PRB protein expression in highly differentiated endometrial carcinoma was not significantly reduced , The difference was not statistically significant, the expression was significantly lower in poorly differentiated endometrial carcinoma, the difference was statistically significant. (2) Compared with normal endometrial tissue, the methylation rate of CpG island in PRA promoter region was lower in endometrial carcinoma tissues, the difference was not statistically significant, the methylation rate of CpG island in PRB promoter region was higher, The difference was statistically significant, and the degree of tissue differentiation, and pathological type with or without lymph node metastasis and the presence or absence of myometrial invasion unrelated; endometrial cancer Hec-1-B cell line, before adding ADC ADC unmethylation PRB appeared methylated bands. After adding ADC, the unmethylated bands of PRA remained unchanged and the methylated bands of PRB disappeared. (3) After addition of different concentrations of ADC in Hec-1-B cell line of endometrial carcinoma, PRA protein expression did not change significantly, PRB protein expression increased to varying degrees. [Conclusion] Abnormal methylation of PR gene exists in endometrial carcinoma and Hec-1-B cell lines, which is closely related to the down-regulation of PR expression. The selective methylation of the subtype B promoter of PR gene leads to PRB inactivation, followed by PRB protein expression decreased or disappeared, may be involved in the formation of endometrial cancer. ADC can restore PRB expression through demethylation, which may improve the clinical endocrine therapy effect of endometrial cancer.