HOXA11-AS:6作为ceRNA吸附miR-223-3p调控肠黏膜屏障在克罗恩病发病中的作用研究

Intestinal mucosal barrier dysfunction is a key component in the pathogenesis of Crohn's disease (CD). It is believed that long non-coding RNA (lncRNA) may be one of the novel regulatory targets of CD while the exact mechanisms remain controversial. By RNA sequencing (RNA-seq) of whole transcriptome and analyzing clinical and animal samples, we have recently found that the expression of lncRNA HOXA11-AS:6 was significantly decreased in CD patients and colitis mice model comparing with the normal controls. Also, we have found that HOXA11-AS:6 could positively regulate the expression of CLDN8, a key gene in regulating intestinal mucosal barrier function. Further bioinformatics analysis revealed that HOXA11-AS:6 could specifically bind to miR-223-3p, which has been confirmed to regulate the expression of CLDN8 in CD in our previous studies. Based on aforementioned evidence, we innovatively hypothesize that HOXA11-AS:6 may act as a competing endogenous RNA (ceRNA), decoying miR-223-3p,thus protecting CLDN8 mRNA and rehabilitating intestinal mucosal barrier in CD pathogenesis. To verify the hypothesis, this study aims to use qPCR, western blotting, luciferase test, fluorescence in situ hybridization, cell immunofluorescence, RNA binding protein immunoprecipitation and RNAscope techniques to demonstrate that HOXA11-AS:6 could function as a ceRNA to sponge miR-223-3p and modulate the expression of CLDN8 in vitro cell experiment, "intestine organoid" model and in vivo gene knockout mice model, which thereby regulates the imbalance of intestinal mucosal barrier function in CD. Eventually, the research may confirm a novel pathway of preventing the development of CD and shed new light on treating CD.

黏膜屏障功能障碍是克罗恩病（CD）发病的关键环节；长非编码RNA可能参与其调控，但具体机制未明。我们通过全转录组测序及临床和动物标本验证，发现长非编码RNA HOXA11-AS:6在CD患者及结肠炎小鼠肠黏膜表达均较正常对照下调，并可影响黏膜屏障关键基因CLDN8表达，且两者呈正相关。进一步生物信息学分析发现HOXA11-AS:6可调节miR-223-3p，而后者我们已证实可调控CLDN8表达。由此，我们首次提出HOXA11-AS:6可作为ceRNA吸附miR-223-3p影响CLDN8表达进而调节CD肠黏膜屏障功能。本项目拟通过细胞实验、“微小肠”及基因敲除动物模型，采用qPCR、蛋白印迹、荧光素酶试验、荧光原位杂交、RNA免疫共沉淀及RNAscope等技术阐明HOXA11-AS:6可作为ceRNA吸附miR-223-3p从而调控CD肠黏膜屏障功能的新机制，有望为CD的治疗提供新策略。

黏膜屏障功能紊乱是克罗恩病（CD）发病的关键环节。最近，长非编码RNA（lncRNA）通过调控免疫稳态及黏膜屏障在IBD发病中的发挥重要作用。我们的基因芯片及GEO数据库芯片结果发现，lncRNA HOXA11-AS:6在CD患者结肠黏膜及小鼠DSS结肠炎中表达明显下降，但在CD发病中的具体机制尚未明确。我们扩大样本，证实lncRNA HOXA11-AS:6在CD患者结肠黏膜及小鼠DSS结肠炎中表达明显下调；进一步使用HOXA11-AS:6全身性敲除鼠构建DSS结肠炎模型，发现敲除HOXA11-AS:6可加重结肠炎症，并且肠黏膜CLDN8表达下调。细胞实验亦证明，过表达HOXA11-AS:6使肠上皮细胞屏障功能增强。数据库预测HOXA11-AS:6可能通过ceRNA机制吸附miR-223-3p调控CLDN8表达。进一步体内外实验如荧光素酶基因报告实验、RIP实验验证HOXA11-AS:6通过结合miR-223-3p维持肠道屏障功能及减轻肠道炎症。同时，我们进一步通过类器官技术探索HOXA11-AS:6在CD发病中的作用。我们成功培养出野生型小鼠及Hoxa11os敲除小鼠的结肠类器官，对野生型小鼠及Hoxa11os敲除小鼠的结肠类器官进行测序，发现差异表达基因富集于细胞黏附分子，进一步证明HOXA11-AS:6:6通过调控肠上皮细胞屏障功能在CD中发挥重要作用。同时，我们通过qPCR及WB验证了敲除组小鼠及对照小鼠类器官CLDN8表达，结果显示敲除组小鼠类器官CLDN8表达下降。本项目发现HOXA11-AS:6在维持CD肠道屏障中起重要作用，其主要通过ceRNA机制结合miR-223-3p调控肠上皮细胞CLDN8表达维持肠黏膜屏障功能。选择性促进肠上皮细胞HOXA11-AS:6表达有望成为促进CD黏膜愈合精准治疗的新手段。