HOTAIR与DNMT1相互作用在皮肤细胞衰老中的机制研究

Several studies have demonstrated that long non-coding RNA could regulate aging. Our preliminary experiments showed that expression of lncRNA-HOTAIR increased in the senescent keratinocytes and skin fibroblasts. Senescence phenotype was alleviated dramaticlly after HOTAIR inhibition in cells. At the same time, we also found that HOTAIR repressed DNMT1 expression and interestingly, expression of HOTAIR increased after silence of DNMT1. In our present study, we will firstly explore the role of HOTAIR in the senescence of skin cells. Then, we will investigate the mechanism of how HOTAIR effect on DNMT1 expression and cellular senescence by using RNA pull-down, DNA methylation profiling, et al. Moreover, the regulation of DNMT1 in expression level and methylated level of HOTAIR promoter are to be further studied by bisulfite sequencing. Finally, we will verify the phenomenon above in vivo by establishing transgenic mice. Our study aims to explore the mechanism of HOTAIR-DNMT1 interaction on the senescence of skin cells and provide a new direction and target gene in the research field and therapeutic field of skin aging.

已有研究表明衰老的发生可能受到了长链非编码RNA的调节。我们的预实验发现HOTAIR在自然衰老和传代衰老的皮肤角质形成细胞和成纤维细胞中表达均上升，抑制HOTAIR能减轻细胞的衰老表型；同时，发现HOTAIR能负向调控DNA甲基转移酶1（DNMT1）的表达，且DNMT1沉默后能明显促进HOTAIR的表达。本研究中，我们将先明确HOTAIR在皮肤细胞衰老中的作用，再利用RNA沉降技术及DNA甲基化谱等技术探讨HOTAIR通过DNMT1在细胞衰老过程中的作用机制；再通过重亚硫酸氢盐测序等方法了解DNMT1对HOTAIR表达量及基因启动子甲基化水平的调节，以明确DNMT1对HOTAIR的作用机制；最后利用HOTAIR基因敲除鼠和表皮条件性DNMT1基因敲除鼠，在体内验证上述现象。本研究的最终目的在于明确HOTAIR在皮肤细胞衰老中的作用和机制，为探索皮肤衰老的机制和治疗方法提供新的方向。

已有研究证明DNMT1对于人皮肤成纤维细胞的衰老具有保护作用，但上游调控机制不明。我们的实验发现长链非编码RNA-HOTAIR在人皮肤成纤维细胞的衰老过程中表达上升，且对于人皮肤成纤维细胞的衰老具有保护作用。HOTAIR的RNA沉降和蛋白分离结果均不支持HOTAIR与DNMT1结合，HOTAIR siRNA处理成纤维细胞后也不能明显降低DNMT1的表达，因此重新调整试验方向，探索DNMT1上游的其他调控机制。人来源脐带间充质干细胞外泌体研究对于人皮肤成纤维细胞的光老化过程具有保护作用，而尿源干细胞外泌体无明显的调控作用。脐带间充质干细胞外泌体能直接下调DNMT1的表达，通过外泌体图谱分析显示外泌体主要是通过其中的miR-152来靶向DNMT1从而调控人皮肤成纤维细胞的衰老过程。本研究探讨了HOTAIR以及外泌体-DNMT1信号通路调控人皮肤成纤维细胞细胞衰老的机制，初步探索了人皮肤成纤维细胞中调控DNMT1从而影响其衰老的上游小RNA，为后续的研究提供了重要的数据和基础。