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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 promoter methylation of DNA binding inhibitor 4 (Id4) in cervical cancer. Methods: Pyrosequencing was used to detect the methylation level of Id4 gene in 17 cases of sporadic cervical squamous cell carcinoma and 15 cases of normal cervical tissue. Real-time quantitative PCR was used to detect the expression of Id4 in tissues and cells. Demethylation of cervical cancer cells SiHa with a DNA methyltransferase inhibitor (5Aza-dC) alone and in combination with 5Aza-dC and the histone deacetylase inhibitor trichostatin A (TSA) After SiHa treatment, Id4 expression was measured. Results: The level of Id4 methylation in cervical cancer was significantly higher than that in normal cervical tissue [(65.0 ± 24.7)% vs (21.8 ± 10.2)%, P <0.01]. In cervical squamous cell carcinoma and normal cervical tissue, Id4 mRNA expression was inversely correlated with its promoter methylation (Pearson test, r = -0.011, P <0.01). 5Aza-dC alone reduced the level of Id4 methylation in SiHa cells from 98.23% to about 50%, accompanied by a significant increase (25-fold) in Id4 mRNA levels, whereas methylation level and mRNA expression in TSA alone Not obvious. A further decrease in Id4 methylation (down to 29.54%) with 5Aza-dC and TSA and a further increase (about 35 fold) in its mRNA expression level were observed. Conclusion: The Id4 gene is hypermethylated in cervical cancer and mediates the silencing of Id4 expression. Histone deacetylation and DNA methylation play a synergistic role in the mechanism of Id4 epigenetic silencing. Id4 or as a target of epigenetic therapy.