MEG3作为ceRNA调控脂多糖诱导仔猪单核细胞炎性因子表达的分子机制

Endotoxin / lipopolysaccharide (LPS) is one of the main pathogenic factors that seriously endanger the health of livestock and poultry, which can induce the excessive release of inflammatory factors and lead to systemic inflammatory response. In our previous study, we found that lncRNA MEG3 was significantly up-regulated in liver and peripheral blood mononuclear cells (PBMCs) of the piglets challenged by LPS, indicating it participated in the LPS-induced inflammatory response. Recent studies show that MEG3 can interact with the miRNA as a competing endogenous RNA(ceRNA) to participate in the expression regulation of target genes，which exert an important role in the cancer and inflammation. Therefore, we speculate that MEG3 could function as a ceRNA to regulate LPS-induced mRNA expression of inflammatory factors. In this study, we will treat PBMCs of piglets with LPS to establish cell model of inflammatory response. In the cell model, overexpression/RNAi, cytokine antibody microarray, luciferase assay, RIP, qPCR will be used to clarify the regulatory role of MEG3 on inflammatory factors, and then identify the candidate miRNA interacting with MEG3 and its key target genes, in order to elucidate the mechanism by which MEG3 functions as ceRNA of candidate miRNA to regulate the expression of inflammatory factors. This study is helpful to enrich the regulatory network of inflammatory factors, and provide new ideas to explore the treatment of endotoxin induced inflammatory response.

内毒素/脂多糖(LPS)是目前严重危害畜禽健康的主要致病因子之一，能诱导炎性因子的过度释放导致全身性炎症反应。我们前期发现lncRNA MEG3在LPS刺激后的仔猪肝脏和单核细胞中显著上调，提示其参与LPS介导的炎症反应。近年研究报道，MEG3作为一种竞争性内源RNA(ceRNA)与miRNA相互作用，参与靶基因的表达调控，进而在肿瘤发生和炎症反应中发挥重要作用。为此，我们推测MEG3可通过ceRNA机制调控LPS诱导的炎性因子表达。本项目利用LPS建立仔猪单核细胞炎症模型，通过干扰/过表达、细胞因子抗体芯片、荧光素酶实验、RIP、qPCR等方法，首先明确MEG3对炎性因子的调控作用，并进一步筛选鉴定MEG3互作的候选miRNA及其关键靶基因，以探索MEG3作为ceRNA靶向候选miRNA调控炎性因子表达的机制。本研究有助于丰富炎性因子的调控网络，为探索缓解内毒素引起的炎症反应提供新思路。

本项目以“MEG3作为ceRNA调控脂多糖诱导仔猪单核细胞炎性因子表达”这一科学假设为核心，围绕 i）MEG3与LPS诱导的炎症反应关系；ii）LPS对PBMC细胞中miRNA和lncRNA表达谱的影响；iii）关键炎症相关候选lncRNA在动物水平炎症组织中的表达变化及其调控炎症的分子机制，这三方面的内容进行了积极的探索研究。. 利用LPS刺激仔猪PBMCs细胞诱导炎症反应，在不同时间点检测MEG3表达变化，结果表明MEG3呈上调表达趋势。但由于MEG3在细胞中的表达丰度低，RACE实验无法获得其全长，导致MEG3的功能研究难以实现。基于此，课题组将更多精力放在研究LPS刺激后PBMC细胞中miRNA和lncRNA表达谱变化以挖掘新的线索。课题组按照调整方案实施，获得的关键结果如下：（1）PBMC细胞中表达丰度排名前十的miRNAs；（2）差异表达的15个miRNAs、43个lncRNAs、1082个mRNAs，其中ssc-miR-122、ssc-miR-129b、ssc-miR-17-5p、ssc-miR-152、ENSSSCT00000047491、ENSSSCT00000049770和ENSSSCT00000051636为关键候选基因；（3）生物信息学分析表明差异表达的miRNAs和lncRNAs靶基因主要参与炎症相关信号通路，并构建了lncRN-mRNA的调控网络图；（4）鉴定了3个关键lncRNA的组织表达谱；（5）在LPS诱导的仔猪多个炎症组织中检测3个关键lncRNAs均显著上调；（6）利用0.5μM双氧水（H2O2）和0.5μg/mL呕吐毒素（DON）构建猪肠上皮细胞（IPEC-1）损伤模型，检测lncRNAs-51636和lncRNA-47491均呈现差异表达；（7）获得了重要候选lncRNA的全长序列。本项目首次研究了LPS诱导仔猪PBMC细胞miRNA和lncRNA表达谱变化，并发现了新的关键候选基因为后续研究奠定基础。