猪印记基因簇anti-Rtl1中miRNA的转录机制及其对骨骼肌生长发育的调控作用

A lot of miRNA differentially expressed during the development and growth of porcine skeletal muscle had been identified in our previous works. miRNA located in anti-Rtl1 imprinting cluster with high expression level, many studies revealed that these miRNA play important roles during the development and growth of skeletal muscle, but the transcription control of miRNA expression is one of the least understood aspects of miRNA biogenesis. In our study the expression profile of miRNA (miR-127, miR-136, miR-432, miR-433 and miR-434) will be analyzed in skeletal muscle(30d, 65d, 90d and adult) and satellite cells. Transcription start site will be identified by CAGE-Seq and RLM-RACE; then transcription factor binding site and epigenetic regulation status around promoter region will be examined. Finally, the expression level of miRNA will be quantified after transcription factor overexpression and RNAi for proving the specific activation of miRNA. This project will reveal the regulation and roles of these miRNAs during myoblast growth and differentiation. It will offer important information for further study of the skeletal muscle growth and development in pig and of the promoter evolution among species.

实验室前期测序获得大量猪骨骼肌不同发育时期差异表达miRNA，其中anti-Rtl1簇中的miRNA在骨骼肌中高表达，目前的研究表明这些miRNA对骨骼肌的生长发育具有重要的调控作用，但这些miRNA在猪骨骼肌中的转录调控机制还不清楚。本项目分析了anti-Rtl1簇中5个miRNA在猪不同发育时期以及不同类型骨骼肌和骨骼肌卫星细胞中的表达模式；采用CAGE测序和RLM-RACE技术相结合鉴定miRNA转录起始位点；获得猪anti-Rtl1基因簇的启动子区域后，生物信息学和分子生物学技术相结合分析影响miRNA表达的转录因子以及表观遗传修饰，并通过转录因子过表达和抑制实验验证其对miRNA的调控作用，深入揭示anti-Rtl1簇中miRNA在骨骼肌发育过程中的作用。本项目将为研究miRNA参与骨骼肌生长发育分子机制奠定基础，也为转录因子的基因组进化提供理论参考。

Anti-RTL1簇中的miRNAs在骨骼肌中高表达，已有的研究表明这些miRNAs对骨骼肌的生长发育具有重要的调控作用。在本项目的资助下，我们获得了一些新的结果。. 首先，对Anti-RTL1簇中的5个miRNAs在猪不同组织器官中的表达情况进行分析，结果发现这5个miRNAs在不同的组织器官中的表达趋势一致，miR-431的表达量最低，miR-433的表达量最高；两种不同的转录方法发现5个miRNAs成熟体和前体的表达趋势一致，因此推断这5个miRNAs来自同一转录本。然后分别通过3’和5’RACE获得mmu-miR495基因的3’端452bp和5’端108bp的序列。Chip-seq后EMSA验证p65在miR-98调控区域的结合位点。最后，通过检测DIO3基因的甲基化模式与组织器官表达谱，判断DIO3的组织器官表达不受甲基化的调控。总之，在本项目的资助下，申请人及其小组成员对Anti-RTL1簇中的5个miRNAs的表达调控模式进行了分析，为深入揭示anti-Rtl1 簇中miRNA 在骨骼肌发育过程中的分子机制奠定基础。