NamiRNA-193b-3p靶向IGF2BP1调控山羊骨骼肌卫星细胞增殖分化的分子机制

Increasing studies have shown that miRNAs play an important role in regulating muscle cell proliferation and differentiation. Usually, miRISC represses genes expression in cytoplasm, but recent study has defined a new type of NamiRNA, those miRNAs can promote gene expression. Interestingly, we have found that miR-193b-3p had the characteristics of NamiRNAs. At the meantime, our study has shown that miR-193b-3p could promote IGF2BP1 expression, and it also could promote the proliferation and differentiation of goat skeletal muscle satellite cells. Therefore, we take miR-193b-3p as the core research content, which expresses highly during muscle forming stage. By using Crispr/Cas9 and RNA-seq technologies, we are mainly focusing on the mechanism of NamiRNAs miR-193b-3p activating IGF2BP1, and trying to build miR-193b-3p—IGF2BP1 regulation networks. In this way, we are expecting to explore deeply how miR-193b-3p and IGF2BP1 regulating muscle cell proliferation and differentiation. The molecular mechanism of NamiRNA-193b-3p targeting IGF2BP1 will have an important role in epigenetic regulation of muscle, and also have important theoretical significance for revealing molecular mechanism of muscle growth and development as well.

大量研究表明，miRNAs在肌细胞增殖分化调控中发挥重要作用。miRNA通常以沉默复合物形式在细胞质中抑制基因表达，但近期的研究表明一种新定义的NamiRNA具有激活基因表达的作用。申请人前期研究发现，miR-193b-3p符合NamiRNA特征，同时miR-193b-3p能靶向促进IGF2BP1表达调控山羊骨骼肌卫星细胞的增殖分化。本项目以miR-193b-3p为核心研究对象，综合利用Crispr/Cas9、RNA-seq等技术，重点研究miR-193b-3p促进IGF2BP1表达这一NamiRNA激活基因表达新机制，通过构建miR-193b-3p—IGF2BP1 的调控网络，以全面解析两者调控肌细胞增殖分化的分子机制。本项目的NamiRNA-193b-3p调控IGF2BP1机制将代表肌肉组织表观遗传的又一重要层次，对于深入解析肌肉生长发育的分子机制具有重要意义。

肌肉的生长调控机理一直是肉羊分子育种的核心研究内容。研究表明，miRNA在肌细胞的增殖分化中发挥重要作用。项目前期发现miRNA-193b-3p能调控肌细胞生长，与miRNA经典的负调控基因机理相悖，miR-193b-3p靶向促进IGF2BP1表达。为深入探究山羊miRNA-193b-3p正向调控IGF2BP1的分子机理，本项目研究发现山羊miR-193b-3p在细胞核和细胞质均有分布，其序列具有增强子活性，不仅能正向调控靶基因IGF2BP1表达，还能促进邻近基因（MKL2、PARN 和BFAR）转录，是一个NamiRNA。但miR-193b-3p调控基因表达及肌细胞增殖具有种属特异性，在小鼠成肌细胞 C2C12 中则负调控IGF2BP1及邻近基因 MKL2、PARN 和 BFAR 表达，还会抑制肌细胞增殖并减少细胞G2 期比例。为进一步探究哪些基因受到山羊 NamiRNA193b-3p的正向调控，在山羊肌细胞中过表达miR-193b-3p后进行mRNA-seq，共计筛选471个差异mRNA，其中上调264个，下调207个。对上调基因进行GO富集分析，发现富集于细胞生长和周期有关通路中，其中包括胰岛素样生长因子结合通路，而IGF2BP1表达差异最显著。为了验证IGF2BP1功能，在山羊肌细胞中过表达IGF2BP1后进行RNA-seq，生物信息学分析后筛选得到59、49、36和44个差异表达lncRNA、circRNA、miRNA及mRNA，并以此为基础构建了IGF2BP1相关的分子调控网络。最后，分析IGF2BP1下游调控的基因，发现其能显著促进novel circRNA circMYO9B的表达，且circMYO9B能促进山羊肌细胞 的增殖，表明IGF2BP1可通过circMYO9B发挥生物学功能。综合项目研究结果，证明了miR-193b-3p正向调控IGF2BP1分子机理，构建了NamiR-193b-3p/IGF2BP1相关的分子调控网络，初步验证了靶基因IGF2BP1下游调控的circMYO9B功能。