M2型巨噬细胞源性外泌体miR-590-3p调控LATS1/YAP/β-catenin通路促进溃疡性结肠炎黏膜修复的机制研究

M2 phenotype macrophages are involved in tissue repair. However, the mechanisms by which regulate epithelial regeneration in ulcerative colitis have not been fully characterized. Using exosomal-miRNAs microarray analysis, we screened miR-590-3p, which was significantly up-regulated in M2 macrophages-derived exosomes, as our research target. In vitro, co-cultivation assay demonstrated that exosomal-miR-590-3p could transfer to colonic epithelial cells(CECs). Meanwhile, scratch assay showed that exosomal-miR-590-3p promoted the proliferation of CECs. Furthermore, miR-590-3p down-regulated the expression level of LATS1, which is inhibitor of YAP/β-catenin signaling pathway. Thus, we hypothesized that M2 macrophages derived exosomal-miR-590-3p promote the self-renewal and proliferation of CECs by down-regulation of LATS1 and subsequently activating YAP/β-catenin signaling pathway. To identify this hypothesis, we will employ cell and molecule, mice model and human sample to explore the role and molecular mechanism of M2 macrophages derived exosomal-miR-590-3p in the proliferation of CECs, which will offer new clues into the clinical therapy for UC.

M2型巨噬细胞与组织修复相关，但在溃疡性结肠炎（UC）黏膜修复中的作用机制尚不明确。前期工作发现M2型巨噬细胞源性外泌体（M2-exos）可促进肠上皮细胞的增殖。通过外泌体miRNA测序分析，发现M2-exos的差异基因miR-590-3p，体外共培养和划痕实验证实exosomal-miR-590-3p可转运至肠上皮细胞，并可促进细胞增殖。RT-PCR和WB发现miR-590-3p可下调YAP/β-catenin通路的抑制因子LATS1的表达。因此，我们提出假说：M2-exosomal-miR-590-3p可靶向肠上皮细胞，通过下调LATS1表达，激活YAP/β-catenin通路，从而增强该细胞的自我更新及增殖能力，最终促进UC黏膜修复。我们拟从体外细胞、动物模型及临床三个层面探讨M2-exosomal-miR-590-3p调控肠上皮细胞增殖的作用及分子机制，为UC的临床治疗提供新线索。

溃疡性结肠炎是肠道的慢性易复发炎症，与免疫紊乱、菌群失衡、基因易感及环境改变相关，促进粘膜修复达到内镜下的粘膜愈合是溃疡性结肠炎治疗的目标。体外细胞水平研究YAP对巨噬细胞极化的影响，巨噬细胞M2极化对肠上皮细胞增殖的影响，以及巨噬细胞中YAP对巨噬细胞表型转换的作用，更深一步研究 巨噬细胞YAP调节肠上皮细胞增殖的分子机制及Hippo/YAP通路中关键分子间的相互作用。体内动物实验探究巨噬细胞中YAP对肠黏膜损伤修复的作用。临床样本研究巨噬细胞YAP在UC患者疗效检测及预后判断中的意义。本研究首先通过分离小鼠骨髓来源巨噬细胞并诱导为M2型，转染miR-590-3p或空白对照，而后提取外泌体，体内及体外实验验证外泌体处理后肠上皮细胞的增殖及愈合能力，进一步发现M2型巨噬细胞通过外泌体依赖途径促进肠上皮细胞增殖，同时，肠上皮中的YAP介导了M2型巨噬细胞来源外泌体对肠上皮细胞的增殖作用。此外， miR-590-3p在M2型巨噬细胞来源外泌体中显著高表达，可从巨噬细胞转运至肠道上皮细胞中，增强肠道上皮细胞的增殖和修复。 机制上，miR-590-3p通过结合LATS1转录调控区抑制LATS1表达，继而激活YAP/β-catenin转录过程促进肠上皮细胞伤口愈合能力。MiR-590-3p可抑制抑炎因子如TNF-a，IL-Iβ和IL-6。更为重要的是，相对于注射M2巨噬细胞外泌体的DSS小鼠，抑制M2型巨噬细胞中的miR-590-3p导致了DSS小鼠严重的粘膜损伤及粘膜愈合。本研究表明：M2巨噬细胞源性外泌体中的miR-590-3p通过LATS1抑制YAP/β-catenin信号减轻炎症信号、促进肠上皮修复，为UC的临床治疗提供新的研究思路。