Xie Hong-Fu, Liu Ying-Zi, Du Rui, Wang Ben, Chen Meng-Ting, Zhang Yi-Ya, Deng Zhi-Li, Li Ji
Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China.
Key Laboratory of Organ injury, Ageing and Regenerative Medicine of Hunan Province, Changsha, China.
Cell Death Dis. 2017 Mar 9;8(3):e2663. doi: 10.1038/cddis.2017.75.
Skin aging is a complicated physiological process and epigenetic feature, including microRNA-mediated regulation and DNA methylation, have been shown to contribute to this process. DNA methylation is regulated by DNA methyltransferase, of which DNA methyltransferase 1 (DNMT1) is the most abundantly known. But evidence supporting its role in skin aging remains scarce, and no report regards its specifical upstream-regulating molecules in the process of skin aging so far. Here, we found that DNMT1 expression was markedly higher in young human skin fibroblasts (HSFs) than that in passage-aged HSFs, and DNMT1 knockdown significantly induced the senescence phenotype in young HSFs. We predicted the upstream miRNAs which could regulate DNMT1 with miRNA databases and found miR-377 had high homology with a sequence in the 3'-UTR of human DNMT1 mRNA. We confirmed that miR-377 was a potential regulator of DNMT1 by luciferase reporter assays. miR-377 expression in passage-aged HSFs was markedly higher than that in the young HSFs. miR-377 overexpression promoted senescence in young HSFs, and inhibition of miR-377 reduced senescence in passage-aged HSFs. Moreover, these functions were mediated by targeting DNMT1. Microfluidic PCR and next-generation bisulfite sequencing of 24 senescent-associated genes' promoters revealed alterations of the promoter methylation levels of FoxD3, p53, and UTF1 in HSFs treated with miR-377 mimics or inhibitors. We also verified that the miR-377-mediated changes in p53 expression could be reversed by regulation of DNMT1 in HSFs. Similarly, there was a negative correlation between miR-377 and DNMT1 expression in young and photoaged HSFs, HSFs, or skin tissues from UV-unexposed areas of different aged donors. Our results highlight a novel role for miR-377-DNMT1-p53 axis in HSF senescence. These findings shed new light on the mechanisms of skin aging and identify future opportunities for its therapeutic prevention.
皮肤老化是一个复杂的生理过程和表观遗传特征，包括微小RNA介导的调控和DNA甲基化，已被证明与这一过程有关。DNA甲基化由DNA甲基转移酶调控，其中DNA甲基转移酶1（DNMT1）是最为人所知且含量丰富的一种。但支持其在皮肤老化中作用的证据仍然稀少，目前尚无报告涉及其在皮肤老化过程中特定的上游调控分子。在此，我们发现人皮肤成纤维细胞（HSF）中DNMT1的表达在年轻细胞中显著高于传代老化细胞，并且敲低DNMT1可显著诱导年轻HSF出现衰老表型。我们利用微小RNA数据库预测了可调控DNMT1的上游微小RNA，发现miR - 377与人类DNMT1 mRNA 3'-UTR中的一个序列具有高度同源性。通过荧光素酶报告基因检测，我们证实miR - 377是DNMT1的潜在调控因子。传代老化HSF中miR - 377的表达明显高于年轻HSF。miR - 377过表达促进年轻HSF衰老，而抑制miR - 377可降低传代老化HSF的衰老程度。此外，这些功能是通过靶向DNMT1介导的。对24个衰老相关基因启动子进行微流控PCR和新一代亚硫酸氢盐测序发现，在用miR - 377模拟物或抑制剂处理的HSF中，FoxD3、p53和UTF1启动子的甲基化水平发生了改变。我们还证实，在HSF中，DNMT1的调控可逆转miR - 377介导的p53表达变化。同样，在不同年龄供体未暴露于紫外线区域的年轻和光老化HSF、HSF或皮肤组织中，miR - 377与DNMT1表达呈负相关。我们的结果突出了miR - 377 - DNMT1 - p53轴在HSF衰老中的新作用。这些发现为皮肤老化机制提供了新的见解，并确定了其治疗性预防的未来机会。
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