肥胖脂肪细胞外泌体中的miR-223调控Wnt通路促食管上皮肠化生的作用

Visceral obesity is a major risk factor for Barrett’s esophagus(BE). Adipose metabolism disorder induced by visceral obesity is remarkable associated with BE development, however the mechanism is still not clear. It is known that miroRNAs and wnt signal pathway may play key role in the development of BE. In our previous study, the expression of miR-223 was increased in exosomes collected from visceral adipose cells; MiR-223 could regulate normal esophageal cells expressing intestinal proteins to development of BE. APC is the target gen of miR-223, which is related to Wnt/β-catenin signal pathway. Thus, we assume that the mechanism related to obesity in BE is: Visceral obesity adipose cellular exosomes and miR-223 expression increased →active Wnt signal pathway→normal esophageal cells metaplasia to BE. Above this, in this study, we will further extract exosomes from adipose cells from visceral obesity patients and non obesity patients, then verify the expression of miR-223 in these exosomes, normal esophageal squamous epithelia cells and BE cells by qRT-PCR. In Vitro, esophagus squamous epithelial cells are intervened with exosomes 、miR-223、wnt signal pathway activator and inhibitor, after that, APC, Wnt/β-catenin signal pathway, intestinal proteins are detected using qRT-PCR and western-blot. Then miR-223 is transfected into squamous epithelial cells to set up 3-D implantation model in vivo to investigate the relationship between miR-223、APC、Wnt/β-catenin and BE genesis. Therefore, in this study, the mechanism will be explored to develop new strategies for the clinical management of patients with BE, and can be potentially targeted by novel molecular therapies for prevention.

内脏型肥胖是Barrett食管（BE）的高危因素，其导致的脂肪代谢紊乱与BE发生密切相关，但机制不清。已知miRNAs及Wnt通路在BE形成中起重要调控作用。前期实验证明肥胖内脏脂肪细胞外泌体中miR-223表达增加, miR-223上调可促使食管上皮表达肠化生蛋白。生物信息学分析miR-223可通过抑制APC激活Wnt通路。因此，我们假设内脏型肥胖引发BE的机制为：肥胖内脏脂肪细胞外泌体中miR-223表达增加→活化Wnt通路→促食管上皮肠化生形成BE。本研究拟进一步比较内脏肥胖和非肥胖者脂肪细胞外泌体中miR-223的表达；分别用外泌体、miR-223、wnt通路激活剂及阻断剂干预食管上皮细胞，在基因和蛋白水平检测Wnt通路及肠型标志表达变化；并构建3-D食管植入模型探讨其体内机制。本项目旨在探索脂肪代谢紊乱促食管上皮肠化生的机制，为BE的发生机制研究提供新思路，为BE的防治提供新靶点

内脏型肥胖是Barrett食管（BE）的高危因素，其导致的脂肪代谢紊乱与BE发生密切相关，但机制不清。已知miRNAs及Wnt通路在BE形成中起重要调控作用。本研究分为四部分进行，首先通过提取肥胖BE患者血清中外泌体，检测miRNAs表达差异谱，获得差异表达miRNAs共36个，其中6个在BE患者外泌体中低表达，30个高表达状态，其中miR-223-5p表达升高，并通过qRT-PCR验证芯片结果是真实可信的。第二部分在基因水平检测了miR-223-5p在腹型肥胖患者内脏脂肪组织中呈高表达状态，验证了miRNA-223-5p在肥胖内脏脂肪组织中的表达。第三部分验证miR-223有促进正常食管鳞状上皮细胞肠化生的作用。在蛋白和基因水平分别检测了miR-223-5p在BE和正常食管粘膜组织中的表达水平以及其在HEEC和BAR-T细胞中的表达水平。第四部分验证miR-223 可通过靶向 APC 调控 Wnt/β-catenin 信号通路促进正常食管鳞状上皮肠化生。使用Wnt/β-catenin信号通路激动剂Wnt-3a和抑制剂Dkk-1处理HEEC细胞后，通过Western Blot检测BE肠上皮化生标志物蛋白Cdx1/2、Muc2的表达水平。验证APC为miRNA-223-5p的细胞内靶基因之一，通过qRT-PCR和Western Blot验证了在BE组织和HEEC细胞中miR-223-5p表达水平与APC蛋白水平呈负相关，进一步通过双萤光素酶报告基因检测验证了miR-223-5p靶向APC基因。向HEEC细胞分别转染miR-223-5p mimic和miR-223-5p inhibitor，用Western Blot技术检测细胞中肠化生标志物Cdx1、Cdx2和Muc2蛋白的表达水平增高。miR-223-5p可在转录后翻译水平抑制APC蛋白的表达，进而调控Wnt/β-catenin信号通路的活性，影响HEEC细胞中BE肠上皮化生蛋白标志物Cdx1、Cdx2和Muc2的表达。因此，推测内脏肥胖外泌体中miR-223-5p表达上调可能与食管肠化生的发生有关。