表观遗传药物联合诱导口腔癌FMR1NB表达的研究

Epigenetic drug combination induced the expression of FMR1NB in oral carcinoma

目的研究DNA去甲基化药物联合组蛋白去乙酰化酶抑制剂对人口腔癌细胞脆性X智障基因1邻近蛋白(FMR1NB)表达及其启动子甲基化的影响,探寻改善FMR1NB表达异质性的方法和策略。方法DNA甲基化转移酶抑制剂地西他滨(DAC)联合组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)和丙戊酸(VPA)干预人舌鳞癌细胞株Cal27和SCC-9后,采用逆转录-聚合酶链式反应(RT-PCR)、实时定量RT-PCR(qRT-PCR)和蛋白印迹法(Western blot)检测干预前后FMR1NB的表达变化;焦磷酸测序法检测干预前后FMR1NB启动子甲基化的变化。结果与空白对照组相比,DAC及其与TSA和VPA联合组均能显著诱导Cal27和SCC-9中FMR1NB mRNA和蛋白的表达。与DAC单独组比较,Cal27中各联合用药组的FMR1NB mRNA表达水平均显著升高,但FMR1NB蛋白表达无明显变化;而SCC-9中除DAC与TSA联合组不能明显提升FMR1NB mRNA表达水平之外,其余各组均能引起FMR1NB mRNA和蛋白水平的显著升高。此外,两株细胞中FMR1NB mRNA和蛋白表达在三药联合组和各两药联合组之间差异均无统计学意义。进一步甲基化测定显示:除SCC-9的三药联合组之外,其余各给药组在两株细胞中FMR1NB启动子区的整体甲基化水平和所测各CpG位点的甲基化水平均有不同程度地降低。结论DAC及其TSA和VPA联合组普遍可介导FMR1NB启动子去甲基化而增强FMR1NB表达,其中两药联合组的增强表达作用更强。

Objective To investigate the effects of DNA demethylation drugs combined with histone deacetylase inhibitors on fragile X mental retardation 1 neighbor protein(FMR1NB)expression and its promoter methylation in human oral cancer cells and try to find a strategy of weakening the heterogeneity of FMR1NB expression.Methods Human oral cancer cell lines Cal27 and SCC-9 were treated with decitabine(DAC),an inhibitor of DNA methyltransferase,combined with trichostatin A(TSA)and valproic acid(VPA),inhibitors of histone deacetylase.Then reverse transcription-polymerase chain reaction(RT-PCR),quantitative real-time PCR(qRT-PCR)and Western blot were used to detect the expression of FMR1NB and pyrosequencing was used to detect the methylation of FMR1NB promoter.Results Compared with the blank control group,DAC and its combination with TSA and VPA significantly induced the expression of FMR1NB mRNA and protein in Cal27 and SCC-9 cells.Compared with DAC alone group,FMR1NB mRNA expression of each DAC-combined drug groups significantly increased,but FMR1NB protein did not significantly change in Cal27 cells;for SCC-9 cells,except for DAC+TSA group,the mRNA and protein levels of FMR1NB significantly increased in all other groups.In addition,there was no significant difference in the expression of FMR1NB mRNA and protein between the three-combined drugs group and two-combined drugs groups.Further methylation assay showed that the methylation level of the overall FMR1NB promoter and its each CpG site measured were reduced to varying degrees in all treatment groups except for three-combination drug group of SCC-9.Conclusion DAC and its combination with TSA and VPA can enhance the expression of FMR1NB by mediating the demethylation of FMR1NB promoter,wherein the enhanced expression effect of the combination of the two drugs is stronger,suggesting that they have the potential to weaken the heterogeneity of FMR1NB expression and improve the immunotherapy effect of oral cancer.