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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 observe the expression of RAR-β in cervical cancer cells and to explore the relationship between RAR-β DNA methylation and RAR-β gene expression defects. Methods RT-PCR was used to detect the expression of RAR-β mRNA in SiHa, HeLa, C33A and Caski cells. The expression of RAR-β protein was detected by immunofluorescence staining and Western blotting. Methylation Methylation status of RAR-βgene in cervical cancer cells was detected by specific polymerase chain reaction (MSP) and RAR-βgene was detected by 5-deoxy-2’-azacitidine (5-Aza-cdR) DNA methylation status changes, and 5-Aza-cdR treatment of RAR-β gene expression defects. Effect of 5-Aza-cdR on cell proliferation by MTT assay. Results The expression levels of RAR-β mRNA in SiHa, HeLa, Caski and C33A cells were 0.25 ± 0.08,0,0.60 ± 0.19 and 3.12 ± 0.92, respectively. The expression levels of RAR-β were 0.23 ± 0.07,0 and 0.14 ± 0.05 , 0.68 ± 0.21. The expression of RAR-β gene was absent or decreased in SiHa, HeLa and Caski cells, while the expression of RAR-β gene was present in C33A cells. MSP technology detected, SiHa, HeLa, Caski cells exist RAR-β gene DNA methylation, while C33A cells are unmethylated state. The expression levels of RAR-β mRNA in SiHa, HeLa, Caski and C33A cells after treatment with 5-Aza-cdR were 1.82 ± 0.59, 2.13 ± 0.62, 1.67 ± 0.43 and 2.95 ± 0.89, respectively. The expression of RAR- 0.69 ± 0.21,0.83 ± 0.29,0.56 ± 0.16,0.64 ± 0.20.5-Aza-cdR before and after treatment, the expression levels of RAR-β mRNA and protein in SiHa, HeLa and Caski cells were significantly different (P < 0.05). There was no significant difference in the expression of RAR-β mRNA and protein in C33A cells (P> 0.05). 5-Aza-cdR treatment can inhibit the proliferation of cervical cancer cells. Conclusion The expression of RAR-β gene plays an important role in the development of cervical cancer. Abnormal methylation of RAR-β gene is an important reason for the defective expression of this gene.