宫颈癌细胞系中RASSF2A基因启动子区甲基化状态的研究

目的拟通过检测4株不同宫颈癌细胞系HeLa、CaSki、SiHa和C33A中RASSF2A基因启动子区甲基化水平,以及分析去甲基化前后RASSF2A基因mRNA和蛋白表达水平、甲基化状态及细胞生物活性的变化,探讨宫颈癌发生的可能机制及治疗对策。方法采用实时定量聚合酶链反应方法检测去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-dC）作用前后RASSF2A基因的表达;甲基化特异性PCR（MSP）法检测4株宫颈癌细胞系中RASSF2A启动子区甲基化状态;免疫细胞化学方法检测RASSF2A基因表达变化,并用MTT法和流式细胞术观察细胞生物活性的变化。结果宫颈癌细胞系HeLa和SiHa表现为完全甲基化状态,细胞系CaSki和C33A表现为部分甲基化状态。5-aza-dC可使宫颈癌细胞系部分去甲基化,并使RASSF2A基因在宫颈癌细胞中mRNA和蛋白水平表达均显著升高。通过对宫颈癌细胞系HeLa的生物学行为检测发现,通过5-aza-dC的诱导作用,其增殖能力和抗凋亡能力明显下降。结论甲基化是导致宫颈癌细胞中RASSF2A基因表达缺失或降低的重要原因之一。去甲基化药物5-aza-dC能够逆转宫颈癌细胞系RASSF2A基因启动子异常甲基化水平,提高RASSF2A基因的mRNA和蛋白表达水平,提示RASSF2A基因的甲基化水平可以作为评估去甲基化干预是否有效的分子学标志物,为宫颈癌的临床治疗提供新的分子靶点。

Study on the status of RASSF2A gene promoter methylation in cervical cancer cell lines

Objective To explore the possible mechanism and treatment countermeasure of cervical cancer by detecting promoter hypermethylation level of RASSF2 A in four cervical cancer cells（HeLa,CaSki,SiHa and C33A） and analyzing the changes of mRNA and protein level of RASSF2 A,methylation status and biological activity before and after demethylation. Methods Real-time quantitative-polymerase chain reaction（RQ-PCR）was used to examine the expression of RASSF2 A in cervical cancer cells before and after 5-aza-dC treatment. The promoter methylation of RASSF2 A in the four cervical cells was detected by methylation specific PCR（MSP） methods. The expression changes of RASSF2 A was detected by immunohistochemistry,and its influence on biological activity was observed with MTT and flow cytometry. Results The expression of HeLa and SiHa in cervical cancer lines were complete methylation,and the CaSki and C33 A were partial methylation. The 5-aza-dC treatment could make part cervical cancer cells demethylation,as well as increase both the protein expression level and mRNA level of RASSF2 A in cervical cancer cells. Biological behavior detection showed that the proliferation capacity and apoptosis rate of HeLa cell of cervical cancer decreased under the induction of 5-aza-dC. Conclusion The methylation is one of great reasons for expression loss or decline of RASSF2 A in cervical cancer cells. The 5-aza-dC can reverse RASSF2 A gene promoter abnormal methylation level in cervical cancer cells,improve the expression level of mRNA and protein of RASSF2 A gene,which show that the methylation level of RASSF2 A,as a molecular marker,can evaluate whether the demethylation intervention is effective,and provide a new molecular target for clinical treatment of cervical cancer.