HDAC2通过LncRNA-H19调节角质形成细胞EMT/MET参与创面再上皮化的分子机制研究

Keratinocyte (KC) EMT plays an important role in wound re-epithelialization, a key process of normal wound healing, however hyperproliferative and nonmigratory Keratinocyte shows in the Margin area of chronic wounds, which impairs wound re-epitheliazation, but its molecular mechanism remains largely unclear. Our preliminary studies demonstrated that HDAC2-knockout KC cells showed increased migration and EMT features, such as decreased E-cadherin and increased Zeb1. It was found that LncRNA-H19, a positive regulator of EMT, was significantly upregulated in HDAC2-knockout KC cells. On the other hand, the special and temporal expression of HDAC2 during wound healing was proved to be regulated by TGF-β, a factor known to be involved in wound healing, re-epithelialization, and EMT. So, we speculated that, HDAC2 may negatively regulate EMT of KCs by downregulating H19, and together with TGF-β, mediate spatial and temporal regulation of EMT/MET of KCs to promote wound re-epithelialization and wound healing. To confirm this hypothesis, this study intends to investigate how the TGF-β-HDAC2-H19 signaling axis regulate EMT of KCs to mediate wound re-epithelialization, which shall expand our understanding of wound epithelization and inspire new treatment strategy.

KC细胞介导创面再上皮化是创面正常愈合的关键环节，而难愈创面创缘KC细胞常呈现过度增殖，但移行和上皮化障碍，目前机制不清。前期预实验证实，HDAC2敲除的KC细胞迁移增加,呈现EMT表型，EMT标记分子E-钙粘蛋白表达下降，Zeb1表达上调，同时EMT正性调控分子H19表达显著上调。另外，HDAC2于创面愈合过程中呈时空性表达，并受TGF-β（创面愈合、上皮化及EMT的重要调控分子）所调控。为此，我们提出"HDAC2通过直接阻遏H19的表达，负性调控KC细胞EMT，并协同TGF-β信号时空性介导KC细胞的EMT/MET进程，有效调控创面再上皮化和愈合"的机制假设。为证实此假设，本研究拟应用HDAC2敲除及GFP标记的KC细胞，从细胞和动物层面通过系列生物学实验，探索TGF-β-HDAC2-H19信号轴对KC细胞EMT/MET和创面再上皮化的作用机制，为发展难愈创面的治疗新策略提供理论依据。

KC细胞介导创面再上皮化是创面正常愈合的关键环节，明晰其分子调控机制对于难愈创面治疗有重要的理论指导意义。在本项目研究中，我们发现创面微环境中的TGF-β可以通过TGF-β/smad信号通路正向调控KC细胞HDAC2的表达，并促进上皮间质转化（EMT）的发生。进一步地，我们发现敲除/敲低HDAC2可以促进细胞EMT的发生。通过全转录组测序筛选及验证，我们发现LncRNA H19是HDAC2调控EMT发生的下游靶基因。在机制研究中我们发现，HDAC2通过结合转录因子SP1并催化组蛋白H3K27的去乙酰化修饰抑制LncRNA H19的表达，而H19则通过吸附MiR-22-3P并上调MMP14的表达促进EMT的发生。此外，在本项目的资助下，我们还发现：①长链非编码RNA LncDC对于KC细胞的分化有促进作用；②细胞程序性死亡-配体1(PD-L1)通过调控巨噬细胞的极化促进创面愈合。我们的研究揭示了TGF-β/HDAC2/H19信号轴对上皮间质转化的调控作用及机制，并发现Lnc-DC及PD-L1分别对于KC细胞功能和创面愈合的重要调控作用，为发展难愈创面的治疗新策略提供理论依据。