The Role of sEV-associated miR-146a in Tissue Repair

摘要 皮肤创伤是一个重大的医疗保健问题，自1997年以来，没有治疗慢性伤口的方法 已获得FDA批准。造成这种治疗障碍的原因是伤口愈合过程的复杂性。 以及这些活动如何在慢性伤口中被失调和利用的研究相对较少的领域 和细胞转化。我们以前的工作证明，钙粘附素，桥粒芯糖蛋白2(Dsg2)是一种 多效性蛋白，调节肿瘤发生和迁移过程中的有丝分裂信号和亲迁移编程 伤口愈合。我们已经证明，Dsg2在慢性伤口中下调，并且构成 Dsg2在表皮中的过度表达可促进伤口愈合、过度增殖和肿瘤 发展。强调角质形成细胞动力学改变对许多 皮肤病包括慢性伤口、牛皮癣和鳞状细胞癌。DSG2能够 通过其下游调控调节角质形成细胞的增殖和迁移等活动 信号分子包括促增殖和迁移蛋白：EGFR和c-Src。除 对于蛋白质的调节，我们的实验室还证明了Dsg2调节细胞内和小细胞外的 囊泡(SEV)转录组。MiRNAseq揭示，Dsg2导致miR-146a的戏剧性耗竭，这是 是炎症反应的有效调节剂，已被证明可能调节4469个基因， 包括miR-155的水平。因此，有趣的是注意到MIR-155的相应增加。 对Dsg2表达式的响应。最近，我们重点确定了Dsg2的上游调控因素，以及 令我们非常惊讶的是，miR-155针对的是Dsg2，这应该会导致它的退化，然而，这些RNAseq 相同的细胞导致实现了lncRNA，Dsg2-AS1的水平也增加了。这很好 证实了lncRNAs可以“海绵”miRNAs并抑制其靶标的降解。这让我们找到了 考虑一种新的调节机制，Dsg2-AS1通过它海绵miR-155，允许急剧增加 在Dsg2表达式中。初步数据表明，这种机制在正常伤口愈合过程中起作用， MiR-155和Dsg2-AS1同时增加后Dsg2的表达增加就是明证 在伤口修复期间。在这个提议中，我们假设控制Dsg2表达的调控环是 在慢性伤口中改变，例如在糖尿病的情况下，这会导致角质形成细胞被扰乱激活 因此伤口闭合效果不佳。因此，本提案的目的是阐明Dsg2的监管 网络及其对角质形成细胞增殖和迁移的影响，并证明SEV- 以这个网络为靶点的被包裹的RNA物种，作为一种在伤口修复中刺激角质形成细胞激活的方式。 这些研究的结果将证明SEV作为一种强有力的治疗方式的可行性。 并有助于从机理上理解伤口愈合。

ABSTRACT Cutaneous wounds represent a significant healthcare issue and since 1997, no treatment for chronic wounds has received FDA approval. Contributing to this treatment desert is the complexity of the wound healing process and the relatively understudied field of how these activities are dysregulated and exploited in chronic wounds and cellular transformation. Our previous work demonstrated that the cadherin, desmoglein 2 (Dsg2) is a pleiotropic protein that modulates mitogenic signaling and pro-migratory programming in both tumorigenesis and wound healing. We have shown that Dsg2 is down-regulated in chronic wounds and that constitutive overexpression of Dsg2 in the epidermis confers increased wound healing, hyperproliferation, and tumor development. Highlighting the contribution of altered keratinocyte dynamics to the pathology of many dermatological conditions including chronic wounds, psoriasis, and squamous cell carcinoma. Dsg2 is able to modulate keratinocyte activities such as proliferation and migration through its regulation of downstream signaling molecules including the pro-proliferative and migratory proteins: EGFR and c-Src. In addition to the modulation of proteins, our lab has also demonstrated that Dsg2 regulates the cellular and small extracellular vesicle (sEV) transcriptome. miRNAseq revealed that Dsg2 induced the dramatic depletion of miR-146a, which is a potent regulator of the inflammatory response and has been shown to potentially regulate 4469 genes, including the level of miR-155. Therefore, it was interesting to note the consequent increase of miR-155 in response to Dsg2 expression. Recently, we have focused on identifying the upstream regulators of Dsg2, and much to our surprise, miR-155 targets Dsg2, which should lead to its degradation, however, RNAseq of these same cells lead to the realization that the level of the lncRNA, Dsg2-AS1, was also increased. It is well established that lncRNAs can “sponge” miRNAs and inhibit the degradation of their targets. This led us to consider a novel regulatory mechanism whereby Dsg2-AS1 sponges miR-155, allowing for a dramatic increase in Dsg2 expression. Preliminary data demonstrates that this mechanism is at play during normal wound healing, as evidenced by the increase in Dsg2 expression after the concomitant increase of miR-155 and Dsg2-AS1 during wound repair. In this proposal, we hypothesize that the regulatory loop that controls Dsg2 expression is altered in chronic wounds such as in the context of diabetes, which results in perturbed keratinocyte activation and therefore ineffective wound closure. Thus, the goals of this proposal are to elucidate the Dsg2 regulatory network and its impact on keratinocyte proliferation and migration, and to demonstrate the ability of sEV- encapsulated RNA species that target this network as a way to stimulate keratinocyte activation in wound repair. The findings obtained from these studies will demonstrate the feasibility of sEVs as a robust treatment modality and contribute to the mechanistic understanding of wound healing.