LncRNA在早期断奶仔猪肠道损伤过程中的调控作用

Weaning stress causes intestinal injury of piglets, which becomes the biggest obstacle of early weaned technology implemented. Studies showed that early weaning had adverse effects on intestinal structure, digestion and absorption function, and expression of functional proteins. However, the molecular mechanism of damage process of intestine remains unclear. Recently developed long noncoding RNAs (lncRNAs) play an important role in the regulation of gene expression and control of molecular signaling pathways, and are relevant to whole life course and pathologic process. The research of lncRNAs function on piglets’ intestine has not been involved. The project filtered differentially expressed lncRNAs of intestine in early-weaned piglets by high-throughput sequencing, the construction of over-expression and interference vectors, and discovered key lncRNAs and theirs target molecules. Furthermore, we studied the regulation of lncRNAs on target molecules and intestine, and analyzed the location of the lncRNAs in the cell and the proteins interacting with the lncRNAs. The project revealed the regulation role of lncRNAs in the course of intestinal damage, and explored the molecular mechanism of intestinal injury in weaned piglets from a totally new perspective. The successful implementation of the project will have great significance to solve the problem of early weaning and improve the health of piglets.

断奶应激造成仔猪肠道损伤是早期断奶技术实施的最大障碍。尽管研究显示早期断奶对肠道形态结构、消化吸收功能及功能蛋白表达等均产生不利影响，然而仔猪肠道损伤所经历的分子作用机制仍不清楚。最新发展的长链非编码RNA（lncRNA）具有从多个水平调控基因表达，参与支配多种信号通路的重要作用，几乎与机体整个生命进程和病理过程密切相关，而lncRNA与仔猪肠道功能关系研究尚未开展。本项目通过高通量测序、基因过表达和沉默等技术，筛选出早期断奶仔猪肠道差异表达的LncRNA，确定具有关键作用的lncRNA及其靶基因，研究lncRNA对靶基因和肠道的调控功能，分析lncRNA在细胞中的富集定位及与其相互作用的蛋白质，揭示LncRNA在肠道损伤过程中的功能调节，以全新的视角探讨断奶仔猪肠道损伤的分子作用机制。该项目顺利实施对解决仔猪早期断奶难题以及全面提升仔猪健康生产具有重要意义。

断奶应激造成仔猪肠道损伤是早期断奶技术实施的最大障碍，而此时肠道损伤所经历的分子作用机制仍不清楚。最新发展的长链非编码RNA（lncRNA）几乎与机体整个生命进程和病理过程密切相关，但是lncRNA与仔猪肠道功能关系研究尚未开展。基于此，本项目从机体功能代谢和转录组调控两个层面阐述断奶仔猪肠道损伤的作用机制。. 本项目通过对早期断奶仔猪的组织样本、血液及免疫指标等检测，从机体功能代谢方面系统阐述了早期断奶造成仔猪肠道严重损伤（绒毛萎缩、隐窝加深），降低免疫功能（降低IgG、IgM和IgA浓度）及机体抗氧化功能（影响SOD和GSH-PX活性），显著改变了机体生长因子水平（GH、TGF-a和EGF等），降低了仔猪生产性能。从转录组调控方面，本项目对断奶仔猪肠道进行RNA-seq高通量测序，首次确定了仔猪肠道表达lncRNA为3421 个，其中新发现的lncRNA为1612个。在仔猪断奶第4天和第7天，肠道差异表达的lncRNA分别为331和163个，GO和KEGG富集分析发现共有32个GO项极显著富集，涉及相关的lncRNA靶基因有247个,主要富集在 MAPK、TNF、PI3K-Akt、Jak-STAT、Wnt等信号通路。通过生物信息学方法分析及实时定量PCR验证，筛选与肠道损伤及修复相关的6个lncRNA，并确定了其相关的靶基因。进一步挑选差异表达的LNC_000884研究其功能作用，本项目成功构建LNC_000884过表达载体，发现肠道上皮细胞JPEC-J2过表达LNC_000884时，可极显著增高靶基因KLF5表达水平；细胞增殖和凋亡试验发现，LNC_000884过表达时能够极显著促进肠道上皮细胞增殖，极显著降低细胞凋亡率，说明靶基因KLF5预测正确，LNC_000884可通过调控KLF5促进仔猪肠道发育，降低肠道损伤，极可能是调控仔猪肠道损伤的关键lncRNA。本研究首次鉴定了早期断奶仔猪肠道lncRNA，确定具有关键作用的lncRNA及其靶基因，揭示LncRNA在肠道损伤过程中的功能调节，从新的视角探讨断奶仔猪肠道损伤的分子作用机制。