DNA甲基化介导Id4在宫颈癌中的表观遗传沉默

DNA Methylation Induces Inhibitor of DNA Binding 4 Expression Loss in Cervix Cancer

目的:探讨宫颈癌中DNA结合抑制因子4(Id4)表达的启动子甲基化调控。方法:采用焦磷酸测序技术对17例散发性宫颈鳞状细胞癌及15例正常的宫颈组织标本进行Id4基因甲基化水平检测。实时荧光定量PCR检测组织和细胞中Id4表达水平。单独使用DNA甲基转移酶抑制剂(5Aza-dC)去甲基化处理宫颈癌细胞SiHa以及联合使用5Aza-dC和组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)去甲基化处理SiHa后,检测Id4表达。结果:Id4甲基化水平在宫颈癌组织中显著高于正常宫颈组织[(65.0±24.7)%比(21.8±10.2)%,P<0.01]。在宫颈鳞状细胞癌组织和正常宫颈组织中,Id4的mRNA表达水平与其启动子甲基化水平呈负相关(Pearson test,r=-0.011,P<0.01)。单独使用5Aza-dC能使SiHa细胞中Id4的甲基化水平由98.23%降至50%左右,同时伴随Id4mRNA水平的显著提高(25倍),而单用TSA组甲基化水平及mRNA表达改变不明显。联合使用5Aza-dC和TSA时Id4甲基化水平的进一步下降(降至29.54%),及其mRNA表达水平的进一步升高(约35倍)。结论:Id4基因在宫颈癌中呈高甲基化状态,并介导Id4的表达沉默。组蛋白去乙酰化与DNA甲基化在Id4表观遗传沉默机制中起协同作用。Id4或可作为表观遗传治疗的作用靶点。

Objective：To investigate the association between promoter methylation of inhibitor of DNA binding 4 （Id4） and its expression in cervix cancer. Methods：The methylation status of Id4 in 17 squamous cervix cancers and 15 normal cervix tissues was examined by pyrosequencing. Id4 mR- NA expression was assessed by quantitative real time PCR. The DNA methly-transferase inhibi- tor 5-aza-2＇-deoxycytidine （5Aza-dC） and histon deacetylase inhibitor trichostatin A （TSA） were used for demethylation treatment of SiHa cells in vitro. Results：Cervix cancers showed higher Id4 methylation level than normal controls [65.0±24.7]% vs. [21.8±10.21%, P〈0.01）, and Id4 mRNA level was negatively correlated with its methylation level in cervix tissues （Pearson test, r =-0. 011, P〈0.01）. Re-expression of Id4 in SiHa cell line was achieved by treatment with 5Aza-dC, whereas a complete restoration of Id4 expression required the synergistic action of TSA, but not with TSA alone. Re-expression was accompanied by the corresponding Id4 promoter demethylation. Conclusion： Id4 is hypermethylated in cervix cancer, which induces Id4 expression loss. Histon deacetylase is also involved in this mechanism.