子宫颈癌细胞中RAR-β基因表达缺陷与其DNA甲基化的关系

Relationship between RAR-β gene expression defect and its methylation

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-cdR)处理后 RAR-β基因 DNA 甲基化状态的变化,以及5-Aza-cdR 处理对 RAR-β基因表达缺陷的影响。四甲基偶氮唑蓝(MTT)比色法测定5-Aza-cdR 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-cdR 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-cdR 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。

Objective To evaluate the expression of RAR-β 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-β DNA. Methods The expression of mRNA and protein of RAR-β 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-β gene. The demethylating agent 5-aza-2＇-deoxycytidine （5-Aza-cdR） was used to treat methylated cell lines and the change of RAR-β gene methylation and RAR-β 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-β 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-β 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-β gene methylation in SiHa, HeLa and Caski cell lines, while there was no RAR-β gene methylation in C33A cell line. After treated with 5-Aza-cdR, the mRNA and protein expression levels of RAR-β 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-β 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-β gene expression defects play an important role in the carcinogenesis of cervical cancer. Aberrant methylation in promotor region of RAR-β gene may be an important mechanism for the loss of expression of RAR-β gene.