1，25-二羟维生素D3对HaCaT细胞增殖活性及基因组DNA和增殖相关基因启动子甲基化水平的影响

Effect of 1, 25（ OH）2D3 on the proliferative ability of and methylation levels of genomic DNA and proliferation- associated gene promoter in human HaCaT keratinocytes

目的探讨1，25-二羟维生素D3[1，25（OH）2D3]对人表皮角质形成细胞株HaCaT细胞增殖活性、基因组DNA总体甲基化水平及增殖相关基因启动子甲基化水平的影响。方法10^-6、10^-7、10^-8mol／L1，25（OH）2D3作用HaCaT细胞24h后，噻唑蓝（MTF）法检测不同浓度1，25（OH）2D3对HaCaT细胞增殖活性的影响；总体DNA甲基化试剂盒检测基因组DNA总体甲基化水平。10mmol／L1，25（OH）2D3作用HaCaT细胞24h后，实时定量聚合酶链反应（PCR）法检测DNA甲基转移酶（DNMT）和甲基结合蛋白（MBD）mRNA表达水平，甲基化特异性PCR（MS—PCR）检测凋亡相关基因程序化细胞死亡因子5（PDCD5）和基质金属蛋白酶组织抑制因子2（TIMP2）启动子区DNA甲基化状态。结果与溶媒对照组比较，10^-6mol／L1，25（OH）2D3处理组HaCaT细胞增殖活性及基因组DNA总体甲基化显著降低（细胞活力：0．152±0．027比0．290±0．017，P〈0．01；总体甲基化：0．187±0．071比0．316±0．049，P〈0．05），DNA甲基转移酶DNMT3a和DNMT3bmRNA水平显著降低（P值〈0．01或0．05），甲基结合蛋白MECP2和MBD2、凋亡相关基因PDCD5、TIMP2mRNA水平显著升高（P值〈0．01或0．05）。此外，MS—PCR结果显示，1，25（OH）2D3处理组PDCD5、TIMP2基因启动子区DNA甲基化水平（0．380±0．135，0．460±0．172）较溶媒对照组（0．720±0．121，0．680±0．133）显著降低（均P〈0．05）。结论1，25（OH）：D，可降低HaCaT细胞基因组DNA总体甲基化水平，调控DNA甲基化修饰基因表达。此外，1,25（OH）2D3可降低凋亡相关基因PDCD5、TIMP2特异性启动子区甲基化水平，诱导PDCD5、TIMP2基因表达增加，抑制HaCaT细胞增殖。

Objective To estimate the influence of 1, 25（OH）2D3 on the proliferative ability of and methylation levels of genomic DNA and proliferation-associated gene promoter in human HaCaT keratinocytes. Methods Some cultured HaCaT cells were treated with 1, 25 （OH）2D3 of 10-6, 10-7 and 104 mol/L for 24 hours, then, methyl thiazolyl tetrazolium （MTF） assay was carried out to evaluate the proliferative activity of cells, and a global DNA methylation quantification kit was used to determine the global DNA methylation level. Real-time PCR was conducted to quantify the mRNA expression of DNA methyl transferases （DNMTs） and methyl-DNA binding domain （MBD） proteins, and methylation-specific PCR （MS-PCR） to evaluate the methylation status of promoter region in the programmed cell death 5 （PDCD5） and tissue inhibitor of metalloproteinase 2 （TIMP2） genes, in HaCaT cells after 24-hour treatment with 1, 25 （OH）2D3 of 10-6 mol/L. The HaCaT cells receiving no treatment served as the control. Results Compared with the untreated HaCaT cells, those treated with 1,25（OH）2D3 of 10-6 mol/L showed significantly down-regulated proliferative activity （0.152 ±0.027 vs. 0.290± 0.017, P 〈 0.01 ）, global DNA methylation level （0.187 ± 0.071 vs. 0.316 ± 0.049, P 〈 0.05）, DNMT3a and DNMT3b mRNA expression levels （P 〈 0.01 or 0.05 ）, but markedly upregulated mRNA expression levels of MECP2, MBD2, PDCD5 and TIMP2 （P 〈 0.01 or 0.05）. Moreover, the DNA methylation levels within the promoter region of PDCD5 and TIMP2 genes were significantly lower in HaCaT cells treated with 1, 25 （OH）zD3 of 10-6 mol/L than in the control ceils （0.38 ±0.135 vs. 0.72±0.121, 0.46 ± 0.172 vs. 0.68± 0.133, both P 〈 0.05）. Conclusions 1, 25（OH）2D3 may down-regulate the global genomic DNA methylation level of, and modulate the expression of DNA methylation- modifying genes in, HaCaT cells. Furthermore, 1, 25 （OH）2D3 can decrease the promoter methylation levels but induce the overexpression of PDCD5 and TIMP2 genes, and decelerate the proliferation of HaCaT cells.