鸡miR-122的表达调控及其对肝脏脂类代谢的功能研究

microRNAs(miRNAs) are a class of ～22 nt small noncoding RNAs, post-transcriptionally regulating genes by binding to specific sites in the 3′-UTR of mRNAs. Liver is an important organelle for lipid metabolism in chicken. Many genes including miRNAs are involved in the regulation of lipid metabolism.miR-122 is the most abundant microRNA in the liver. In this study, the RACE method will be used to obtain full length sequence of pri-miR-122, and the bioinformatics,reporter gene assay，chromatin immunoprecipitation(ChIP) and electrophoretic mobility shift assay (EMSA) will be combined to make clear of the structure of miR-122 promoter and the regulatory mechanism of miR-122 gene. Using anti-miR-122 to knockdown the level of miR-122 in chicken hepatocytes, and using RNA-Seq method, the changed expression of genes in hepatocytes will be detected. Combining upregulated genes and predicted target genes of miR-122 in silico, the potential target genes will be screened. Several potential target genes will be chosen to be validated using dual luciferase reporter system and Western Blotting, and the function of miR-122 in lipid metabolism of chicken will be analyzed, considering the functions of target genes. The regulatory mechanism of lipid metabolism and fat deposition of chicken will be explored from another angle in this study.

miRNA主要通过与靶基因mRNA的3'-UTR区结合来实现转录后调控。鸡肝脏是脂类代谢的重要场所，脂类代谢过程涉及了许多基因，也受到miRNA的调控。miR-122是肝脏表达量最高的miRNA，本研究用RACE的方法，获得鸡pri-miR-122的全长序列，并结合生物信息学和报告基因的方法手段、ChIP和EMSA技术，弄清鸡miR-122基因的启动子结构和表达调控机制；同时利用转染Anti-miRNA的方法，使鸡肝脏细胞中miR-122的量下降，然后通过RNA-Seq技术检测细胞中基因表达的改变情况，结合表达上调的基因和生物信息学方法预测的miR-122靶基因来确定鸡miR-122的候选靶基因，再利用体外双荧光素酶报告系统和Western Blotting等验证其中的部分靶基因，结合靶基因的功能来分析miR-122在鸡脂肪代谢中的作用，从另一角度探讨鸡脂类代谢和脂肪沉积的调控机理。

本课题的研究目的是弄清鸡miR-122基因的转录起始位点、启动子结构及miR-122表达调控的机理；对miR-122靶基因研究，阐明miR-122在鸡肝脏脂类代谢中的功能。运用qRT-PCR检测发现miR-122在肝脏中表达量最高，在肝脏生长发育过程中上调。采用生物信息学和5’RACE方法确定了鸡miR-122的转录起始位点位于pre-miR-122上游3294bp位置，用序列删除技术证明其核心启动子区位于pre-miR-122上游3.8~3.2 kb区域，其启动子区含有转录因子HNF3β的结合位点，点突变后能显著降低启动子活性，用EMSA证明HNF3β能与鸡miR-122启动子区结合，推测HNF3β能结合该位点激活miR-122的转录。用生物信息学方法预测到364个鸡miR-122的靶基因，挑选与肝脏代谢相关的预测靶基因P4HA1、PKM2、TGF-β3、FABP5、ARCN1、VNN1和BZW2，用体外双荧光素酶报告系统和靶位点突变实验证明这7个基因及预测靶位点分别是miR-122的靶基因和靶位点。转录组测序发现原代鸡肝细胞miR-122抑制后共有123个基因差异表达，其中64个基因表达显著上调，59个基因表达显著下调；GO分析显示21个基因参与鸡肝脏代谢过程，表明miR-122可能通过影响这些基因而调控鸡肝脏代谢。测序及qRT-PCR结果发现miR-122抑制后靶基因P4HA1、PKM2、TGF-β3、VNN1和BZW2 mRNA表达水平上调，FABP5下调，而ARCN1不受影响，Western Blotting分析表明P4HA1蛋白上调，进一步说明miR-122可调控这些靶基因。使用RNAi干扰原代鸡肝细胞中TGF-β后发现其通路下游基因ACTA2表达上调，LEF1和CCNB2表达下调，表明miR-122通过调控TGF-β3可能影响这些基因进而调控肝脏代谢。