DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts
DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts
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发表时间：
2019-07
期刊：
影响因子：
2.2
通讯作者：
Hitomi Yonemura;Akiko Futakuchi;Miyuki Inoue-Mochita;Tomokazu Fujimoto;Eri Takahashi;H. Tanihara;Toshihiro Inoue
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文献类型：
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作者：
Hitomi Yonemura;Akiko Futakuchi;Miyuki Inoue-Mochita;Tomokazu Fujimoto;Eri Takahashi;H. Tanihara;Toshihiro Inoue
目的探讨DNA甲基转移酶抑制剂对人结膜成纤维细胞(HConF)纤维化的影响。方法用甲基转移酶抑制剂5-aza-2‘-脱氧胞苷(5-aza-2’-deoxcytidine，5-aza-DC)处理HConF 48h，传代后加入5 ng/ml转化生长因子-β2 48h，用免疫印迹法检测α-平滑肌肌动蛋白(α-SMA)、细胞外基质蛋白和磷酸化Smad3的表达水平。第一代后，将COL1A2启动子与荧光素酶基因的融合载体导入HConF，通过荧光素酶报告基因检测其活性。结果5-aza-DC(0.1、1.0、10βM)可剂量依赖性地抑制转化生长因子-SMA-2诱导的α-SMA上调。10μM 5-aza-DC可抑制I型胶原的表达。相反，5-aza-DC对纤维连接蛋白或磷酸化Smad3的表达没有抑制作用。但5-aza-DC可抑制COL1A2启动子的活性。结论DNA甲基转移酶抑制剂可部分抑制HConF的纤维化改变，提示该抑制剂对HConF中COL1A2启动子有间接抑制作用。
Purpose This study aimed to clarify the effects of a DNA methyltransferase inhibitor on fibrogenetic changes in human conjunctival fibroblasts (HConF). Methods HConF were pretreated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) for 48 h. After one passage, the cells were treated with 5 ng/ml of transforming growth factor (TGF)-β2 for 48 h, and the expression levels of α-smooth muscle actin (α-SMA), extracellular matrix proteins, and phosphorylated Smad3 were evaluated with western blotting. A fusion construct between the COL1A2 promoter and the luciferase gene was introduced into the HConF after the first passage, and the construct’s activity was detected via a luciferase reporter gene assay. Results TGF-β2-induced upregulation of α-SMA was suppressed by pretreatment with 5-Aza-dC (0.1, 1.0, and 10 μM) in a dose-dependent manner. Upregulation of type I collagen was also suppressed by 10 μM 5-Aza-dC pretreatment. In contrast, 5-Aza-dC had no inhibitory effect on the expression of fibronectin or phosphorylated Smad3. However, COL1A2 promoter activity was suppressed with 5-Aza-dC pretreatment. Conclusions In HConF, fibrogenetic changes were partly suppressed with a DNA methyltransferase inhibitor, suggesting an indirect inhibitory effect of the inhibitor on the COL1A2 promoter in HConF.