子宫颈癌细胞中RAR-β基因表达缺陷与其DNA甲基化的关系

目的观察宫颈癌细胞中RAR-β基因表达的情况,并探讨RAR-β基因DNA甲基化与RAR-β基因表达缺陷的关系。方法采用RT-PCR技术检测宫颈癌细胞系SiHa、HeLa、C33A和Caski细胞中RAR-β mRNA的表达,免疫荧光化学染色法及蛋白印迹法检测其RAR-β蛋白的表达,同时应用甲基化特异性聚合酶链反应（MSP）技术检测宫颈癌细胞中RAR-β基因DNA甲基化状态及采用5-脱氧-2’-杂氮胞苷（5-Aza-edR）处理后RAR-β基因DNA甲基化状态的变化,以及5-Aza-edR处理对RAR-β基因表达缺陷的影响。四甲基偶氮唑蓝（MTF）比色法测定5-Aza-edR对细胞增殖的影响。结果SiHa、HeLa、Caski和C33A细胞中RAR-β mRNA的表达水平分别为0．25±0．08、0、0．60±0．19、3．12±0．92,RAR-β蛋白表达水平分别为0．23±0.07、0、0．14±0．05、0．68±0．21。SiHa、HeLa、Caski细胞中存在RAR-β基因的表达缺失或低下,而C33A细胞中存在RAR-β基因的表达。MSP技术检测发现,SiHa、HeLa、Caski细胞中存在RAR-β基因DNA甲基化,而C33A细胞为非甲基化状态。5-Aza-edR处理后,SiHa、HeLa、Caski和C33A细胞中RAR-β mRNA的表达水平分别为1、82±0．59、2．13±0.62、1.67±0．43、2．95±0．89,RAR-β蛋白表达水平分别为0．69±0．21、0．83±0．29、0．56±0．16、0.64±0.20。5-Aza-cdR处理前后SiHa、HeLa、Caski细胞中RAR-β mRNA和蛋白的表达水平比较,差异均有统计学意义（P〈0．05）;C33A细胞中RAR-β mRNA和蛋白的表达比较,差异则无统计学意义（P〉0、05）。5-Aza-edR处理后能抑制宫颈癌细胞的增殖。结论RAR-β基因的表达缺陷在宫颈癌的发生中起重要作用,RAR-β基因DNA异常甲基化是导致该基因表达缺陷的重要原因。

Relationship between RAR-beta gene expression defect and its methylation

OBJECTIVE: To evaluate the expression of RAR-beta gene in cervical carcinoma cell lines SiHa, HeLa, C33A and Caski and to analyze the relation between their gene expression and the promoter methylation of RAR-beta DNA. METHODS: The expression of mRNA and protein of RAR-beta gene in the four cell lines were analyzed by RT-PCR, western blot and immunofluorescence, respectively. Methylation specific PCR (MSP) was used to check whether there was methylation in the promoter of RAR-beta gene. The demethylating agent 5-aza-2'-deoxycytidine (5-Aza-cdR) was used to treat methylated cell lines and the change of RAR-beta gene methylation and RAR-beta gene expression defects were observed. The cell proliferation was assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method. RESULTS: The mRNA and protein expression levels of RAR-beta in cell lines SiHa, HeLa, Caski and C33A were 0.25 +/- 0.08, 0, 0.60 +/- 0.19, 3.12 +/- 0.92 and 0.23 +/- 0.07, 0, 0.14 +/- 0.05, 0.68 +/- 0.21, respectively. The mRNA and protein expression of RAR-beta in SiHa, HeLa and Caski cell lines were decreased or silenced, whereas its expression increased in C33A cell line. MSP method showed that there were RAR-beta gene methylation in SiHa, HeLa and Caski cell lines, while there was no RAR-beta gene methylation in C33A cell line. After treated with 5-Aza-cdR, the mRNA and protein expression levels of RAR-beta in SiHa, HeLa, Caski and C33A cell lines were 1.82 +/- 0.59, 2.13 +/- 0.62, 1.67 +/- 0.43, 2.95 +/- 0.89 and 0.69 +/- 0.21, 0.83 +/- 0.29, 0.56 +/- 0.16, 0.64 +/- 0.20 respectively. The mRNA and protein levels of RAR-beta had a significant difference between before and after interference with 5-Aza-cdR in SiHa, HeLa, and Caski cell lines (P < 0.05). However, they had no significant difference between before and after interference with 5-Aza-cdR in C33A cell line (P > 0.05). The 5-Aza-cdR treatment could suppress cell proliferation. CONCLUSIONS: The RAR-beta gene expression defects play an important role in the carcinogenesis of cervical cancer. Aberrant methylation in promotor region of RAR-beta gene may be an important mechanism for the loss of expression of RAR-beta gene.