Fu Shuhao, Sun Li, Zhang Xiaoyan, Shi Huimin, Xu Kang, Xiao Yiqin, Ye Wen
Department of Ophthalmology, Huashan Hospital, Fudan University, Shanghai, China.
Department of Ophthalmology, Huashan Hospital, Fudan University, Shanghai, China.
Exp Eye Res. 2017 Dec;165:47-58. doi: 10.1016/j.exer.2017.09.004. Epub 2017 Sep 9.
The principle reason of high failure rate of glaucoma filtration surgery is the loss of filtration function caused by postoperative scar formation. We investigated the effects of 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA methyltransferases inhibitor, on human Tenon's capsule fibroblasts (HTFs) differentiation and fibrosis and its mechanism of action, especially in relation to transforming growth factor (TGF)-β1 signaling. TGF-β1 was used to induce differentiation of cultured HTFs. 5-Aza-dc suppressed DNA methyltransferases (DNMTs) activity 6 h after treatment with a course corresponding to that of TGF-β1-induced reduction of DNMT activity without affecting cell viability as measured by Cell Counting Kit-8 assay. 5-Aza-dc also reduced DNMT1 and DNMT3a protein expression from 24 to 48 h. HTFs migration evaluated by scratch-wound assay were significantly increased 24 h after 5-Aza-dc treatment, a time course similar to that of TGF-β1. Treatment with 5-Aza-dc significantly increased the mRNA and protein levels of α-smooth muscle actin (α-SMA), collagen-1A1 (Col1A1), fibronectin (FN) and TGF-β type I receptor (TGFβRI). Furthermore, the effects of 5-Aza-dc on DNMT activity suppression, cell migration, and fibrosis were all reversed by a TGFβRI inhibitor- SB-431542. Meanwhile, knockdown of DNMT1 upregulated TGFβRI expression and had the same fibrosis-inducing effect in HTFs, which was also inhibited by SB-431542. Thus, the results indicate that DNA hypomethylation induces HTFs differentiation and fibrosis through up-regulation of TGFβRI. DNA methylation status plays an important role in subconjunctival wound healing.
青光眼滤过手术失败率高的主要原因是术后瘢痕形成导致滤过功能丧失。我们研究了DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-dc）对人Tenon囊成纤维细胞（HTFs）分化和纤维化的影响及其作用机制，特别是与转化生长因子（TGF）-β1信号传导的关系。使用TGF-β1诱导培养的HTFs分化。5-Aza-dc处理6小时后抑制了DNA甲基转移酶（DNMTs）活性，其作用过程与TGF-β1诱导的DNMT活性降低过程一致，且通过细胞计数试剂盒-8检测法测定不影响细胞活力。5-Aza-dc还在24至48小时降低了DNMT1和DNMT3a蛋白表达。通过划痕试验评估，5-Aza-dc处理24小时后HTFs迁移显著增加，这一时期与TGF-β1相似。5-Aza-dc处理显著增加了α-平滑肌肌动蛋白（α-SMA）、胶原蛋白-1A1（Col1A1）、纤连蛋白（FN）和TGF-β I型受体（TGFβRI）的mRNA和蛋白水平。此外，TGFβRI抑制剂SB-431542逆转了5-Aza-dc对DNMT活性抑制、细胞迁移和纤维化的影响。同时，敲低DNMT1上调了TGFβRI表达，并在HTFs中具有相同的促纤维化作用，这也被SB-431542抑制。因此，结果表明DNA低甲基化通过上调TGFβRI诱导HTFs分化和纤维化。DNA甲基化状态在结膜下伤口愈合中起重要作用。
