DNA甲基化和组蛋白乙酰化对人口腔鳞癌细胞中KLF4基因的调控研究

DNA methylation and histone acetylation regulations of KLF4 gene in human oral squamous cell carcinoma cells

目的探讨人口腔鳞癌中DNA甲基化和组蛋白乙酰化对KLF4基因的调控作用。方法体外培养人口腔鳞癌细胞系SCC-6,分别用DNA甲基化抑制剂5-氮-2’-脱氧胞苷(DAC)、组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)处理SCC-6细胞,并据此将SCC-6分为4组,阴性对照组(不做处理)、DAC处理组、TSA处理组、DAC+TSA联合处理组,采用荧光定量PCR方法检测SCC-6细胞经DAC或/和TSA处理前后KLF4 RNA水平的变化;用免疫细胞化学方法检测SCC-6细胞经DAC或/和TSA处理前后KLF4蛋白的变化。结果 SCC-6细胞DAC处理组、TSA处理组、DAC+TSA组的KLF4 RNA水平与阴性对照组相比均有显著升高(P<0.01)。另外,DAC处理组KLF4 RNA水平均高于其他3组(P<0.01)。SCC-6细胞DAC处理组中KLF4蛋白表达明显升高,均高于其他3组(P<0.01)。TSA处理组与DAC+TSA联合组KLF4蛋白表达也都高于阴性对照组(P<0.01);TSA处理组与DAC+TSA处理组间无显著差异(P>0.05)。结论在口腔鳞状细胞癌SCC-6细胞中导致KLF4基因沉默的主要原因可能为DNA甲基化,此外组蛋白乙酰化可能也参与了KLF4表达的调控,但组蛋白乙酰化在提高KLF4表达方面与去甲基化无明显协同作用。

Objective To investigate the regulation of DNA methylation and histone acetylation in oral squamous cell carcinoma（OSCC） cells on KLF4 gene. Methods SCC-6 ceils were divided into four groups, negative group （without any treatment）, DAC group （ treated with DAC- DNA methylation inhibitor）, TSA group （treated with TSA- histone deacetylase inhibitors） and DAC ＋ TSA group（ treated with DAC ＋ TSA）. Quantitative PCR was used to examine the RNA expression of KLF4 in SCC-6 before and after the treatment of DAC or/and TSA. Immuneeytochemistry was used to detect KLF4 protein expression before and after the treatment of DAC or / and TSA in SCC-6 cells. Results KLF4 RNA expression in DAC group, TSA group, and DAC ＋ TSA group was significantly higher than that in the negative control （ P 〈 0.01 ）. There was no significant difference between TSA group and DAC ＋ TSA group （ P 〉 0.05 ）. The KLF4 RNA expression in DAC group was higher than that in other three groups （P 〈0.01 ）. The protein expression of KLF4 in DAC group was significantly higher than that in other three groups. （P 〈 0.01 ）. KLF4 expression was not significantly different between the TSA group and DAC ＋ TSA group （P 〉 0. 05 ）, but was significantly higher in TSA and DAC ＋ TSA groups than that in the negative control （P 〈0.01 ）. Conclusion DNA methylation may be the main reason for KLF4 silence in SCC-6 ceils ,and histone acetylation may also participate in the regulation of KLF4 expression, but DNA methylation and histone acetylation has no synergistic effect on improving the expression of KLF4.