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

摘要 皮肤伤口是一个重要的医疗保健问题，自1997年以来，没有治疗慢性伤口 已获得FDA批准。造成这种治疗沙漠的原因是伤口愈合过程的复杂性 以及这些活动在慢性伤口中是如何失调和利用的相对欠研究的领域 和细胞转化。我们以前的工作表明，钙粘蛋白，桥粒芯糖蛋白2（Dsg 2）是一个 一种多效性蛋白，在肿瘤发生和转移中调节促有丝分裂信号和促迁移程序， 伤口愈合我们已经表明，Dsg2在慢性伤口中下调，并且组成性的 表皮中Dsg2的过表达赋予伤口愈合、过度增殖和肿瘤生长的增加。 发展强调了角质形成细胞动力学改变对许多肿瘤病理学的贡献， 皮肤病，包括慢性伤口、牛皮癣和鳞状细胞癌。DSG2可以 调节角质形成细胞的活动，如增殖和迁移，通过其调节下游 信号分子包括促增殖和迁移蛋白：EGFR和c-Src。除了有 我们的实验室还证明，Dsg2调节细胞和小的细胞外蛋白质， 囊泡（sEV）转录组。miRNAseq揭示Dsg2诱导miR-146a的显著消耗， 是一种有效的炎症反应调节剂，并已被证明可能调节4469个基因， 包括miR-155的水平。因此，有趣的是，注意到miR-155的随之增加， 对Dsg2表达的响应。最近，我们专注于鉴定Dsg2的上游调节因子， 令我们惊讶的是，miR-155靶向Dsg2，这应该导致其降解，然而，这些的RNAseq 相同的细胞导致lncRNA，Dsg2-AS1的水平也增加的认识。公 确立了lncRNA可以"海绵" miRNA并抑制其靶标的降解。这导致我们 考虑一种新的调节机制，其中Dsg 2-AS 1吸收miR-155，从而使其急剧增加 在Dsg2表达中。初步数据表明，这种机制在正常伤口愈合过程中起作用， 如在miR-155和Dsg2-AS1同时增加后Dsg2表达增加所证明的 在伤口修复过程中。在这个提议中，我们假设控制Dsg2表达的调节环是 在慢性伤口中改变，例如在糖尿病的情况下，这导致角质形成细胞活化受到干扰 从而导致无效的伤口闭合。因此，本提案的目标是阐明Dsg 2调节性的作用。 网络及其对角质形成细胞增殖和迁移的影响，并证明sEV- 包封的RNA种类靶向该网络作为刺激伤口修复中角质形成细胞活化的方式。 从这些研究中获得的结果将证明sEV作为一种稳健治疗方式的可行性 并有助于理解伤口愈合的机制。

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.