circSNX25调控猪骨骼肌纤维类型转化的分子机制

Muscle fiber types are regulated by many transcriptional factors and epigenetics. Studies showed that circRNA played an important role in the regulation of gene expression and researches about it mainly focused on the medical field. However, the function and mechanism of circRNA is still unclear and has rare report on livestock and poultry. In our previous work, we identified a novel circRNA (named circSNX25) and found that it was expressed differently between fast and slow muscle which isolated from longissimus dorsi and soleus respectively. In addition, we discovered circSNX25 had a plurality of miRNAs binding sites which also differently expressed in fast and slow muscle. Therefore, we speculate circSNX25 may play an important role in regulating muscle fiber type. In this project, we will synthesize the information of circRNAs, miRNAs transcriptome of different muscle fiber types of skeletal muscle to research the role of circSNX25 in porcine skeletal muscle fiber type, the interaction among circSNX25, candidate miRNAs and its target genes and the influence of circSNX25 on quality. We aim to reveal the role and molecular mechanism of circSNX25 in regulating porcine skeletal muscle fiber type. This study will provide a new view to reveal the role of circRNA in the regulation of skeletal muscle fiber types and a theoretical basis for improving pork quality.

骨骼肌纤维类型受到诸多转录因子及表观遗传学调控。研究发现，circRNA在调控基因表达过程中发挥着重要作用。本项目组研究结果表明，circSNX25在猪慢肌中的表达量显著高于快肌，并且具有多个差异表达miRNA的结合位点。因此，circSNX25可能慢肌纤维形成过程中具有重要作用。目前，有关circRNA的研究主要集中于医学领域，在猪上的研究鲜有报道，特别是对circRNA的功能及机制尚不清楚。本项目拟综合猪不同肌纤维类型骨骼肌的circRNA组、miRNA组和转录组等信息，从细胞水平和个体水平阐明circSNX25对猪骨骼肌纤维类型的调控作用以及circSNX25与候选效应因子及其靶标基因的相互作用，以揭示circSNX25调控猪骨骼肌纤维类型转化的作用及分子机制。本研究不仅为研究circRNA对猪骨骼肌纤维类型的遗传调控机制提供新思路，同时为改善猪肉品质提供科学理论依据。

肌纤维类型受到诸多转录因子及表观遗传学调控。研究表明，circRNA对猪肌纤维类型转化的具有重要作用。本项目对猪背最长肌和比目鱼肌进行高通量测序，筛选鉴定出调控肌纤维类型转化的关键circRNA（circMYLK4）； circMYLK4在猪不同组织中广泛表达；circMYLK4在猪骨骼肌卫星细胞分化后期高表达；circMYLK4主要位于细胞质中；在猪骨骼肌卫星细胞中过表达circMYLK4促进猪慢型肌纤维的分化；12日龄仔猪后肢肌肉注射circMYLK4腺相关病毒4w后，慢肌比例显著升高；通过RNA-pulldown技术，分别筛选出231个与circMYLK4结合的miRNA （miR-423-5p、miR-324he miR-196b-5p等）和51个与circMYLK4结合的蛋白（Sam68和CSRP3等）；采用免疫荧光染色、WB、real-time qPCR、流式细胞术、EdU染色、CCK8等技术，研究结果表明， miR-423-5p 抑制C2C12细胞的增殖与分化；miR-324-5p抑制成肌细胞分化；miR-196-5p促进成肌细胞增殖与分化，且促进成肌细胞向慢肌分化；采用荧光素酶报告实验和RIP等技术，研究表明，circMYLK4部分通过吸附miR196b-5p或者与Sam68结合促进猪快肌纤维向慢肌纤维转化；另外，在细胞和活体水平研究表明，干扰CircNeb促进成肌细胞的增殖与分化，且促进成肌细胞向快肌分化而抑制其向慢肌分化；通过软件预测与荧光素酶报告实验证明，miR-6538可以被CircNeb吸附；miR-6538促进成肌细胞分化，且促进成肌细胞向快肌分化而抑制其向慢肌分化；miR-668-3p通过靶向APPL1抑制成肌细胞的增殖与分化。以上研究结果不仅为研究circRNA在猪骨骼肌纤维类型遗传调控中的作用机制提供新思路，同时为改善猪肉品质提供科学理论依据。