视黄酸受体β基因表达缺陷与其甲基化之间的关系
[Relationship between RAR-beta gene expression defect and its methylation].
作者信息
Gao Yan-ping, Li Min, Zhang Ying-ying, Wang Han, He Xiao-hong, Wang Ze-hua
机构信息
Department of Obstetrics and Gynecology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
出版信息
Zhonghua Fu Chan Ke Za Zhi. 2007 Jul;42(7):472-6.
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.
目的
评估视黄酸受体β（RAR-β）基因在子宫颈癌细胞系SiHa、HeLa、C33A和Caski中的表达情况，并分析其基因表达与RAR-β基因启动子甲基化之间的关系。
方法
分别采用逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（western blot）和免疫荧光法分析四种细胞系中RAR-β基因的信使核糖核酸（mRNA）和蛋白质表达。采用甲基化特异性聚合酶链反应（MSP）检测RAR-β基因启动子是否存在甲基化。使用去甲基化剂5-氮杂-2'-脱氧胞苷（5-Aza-cdR）处理甲基化细胞系，观察RAR-β基因甲基化及RAR-β基因表达缺陷的变化。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法检测细胞增殖情况。
结果
SiHa、HeLa、Caski和C33A细胞系中RAR-β的mRNA表达水平分别为0.25±0.08、0、0.60±0.19、3.12±0.92，蛋白质表达水平分别为0.23±0.07、0、0.14±0.05、0.68±0.21。SiHa、HeLa和Caski细胞系中RAR-β的mRNA和蛋白质表达降低或沉默，而在C33A细胞系中其表达增加。MSP法显示SiHa、HeLa和Caski细胞系存在RAR-β基因甲基化，而C33A细胞系不存在RAR-β基因甲基化。用5-Aza-cdR处理后，SiHa、HeLa、Caski和C33A细胞系中RAR-β的mRNA表达水平分别为1.82±0.59、2.13±0.62、1.67±0.43、2.95±0.89，蛋白质表达水平分别为0.69±0.21、0.83±0.29、0.56±0.16、0.64±0.20。在SiHa、HeLa和Caski细胞系中，5-Aza-cdR干扰前后RAR-β的mRNA和蛋白质水平有显著差异（P<0.05）。然而，在C33A细胞系中，5-Aza-cdR干扰前后无显著差异（P>0.05）。5-Aza-cdR处理可抑制细胞增殖。
结论
RAR-β基因表达缺陷在子宫颈癌发生中起重要作用。RAR-β基因启动子区域的异常甲基化可能是RAR-β基因表达缺失的重要机制。
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