miR-499-5p对猪肌纤维类型转化的调控及其机制研究

Muscle fiber types are closely related to pork quality. Our previous work found that the miR-499-5p level in slow-twitch soleus of pigs was significantly higher than that in fast-twitch extensor digitorum longus, whereas the porcine Sox6 mRNA level was just the opposite. After analysis, we found that the 3'UTR of porcine Sox6 contains eight miR-499-5p target sites with complete complementarity in the seed region. Overexpression of miR-499-5p in mice can cause a complete conversion of fast-twitch muscle fibers to slow-twitch myofibers. We therefore hypothesize that miR-499-5p induces porcine muscle fiber-type transition through inhibition of the expression of its target genes, such as porcine Sox6, and so on. The aim of this project is to make clear whether miR-499-5p can induce porcine muscle fiber-type transition in vitro an in vivo. Based on the identification of porcine Sox6 and other genes screened by iTRAQ quantitative proteomics technology as direct target genes of miR-499-5p, another aim of this project is to elucidate whether miR-499-5p-mediated repression of the expression of its target genes including porcine Sox6 are essential for regulation of porcine muscle fiber-type transition by miR-499-5p in vitro and in vivo. The present project is of importance not only to reveal the mechanisms involved in the control of porcine muscle fiber-type transition, but also to provide theoretical foundation and new strageties for improving pork quality.

肌纤维类型与猪肉品质密切相关。对比猪不同类型骨骼肌中miR-499-5p的表达水平，我们发现比目鱼肌（慢肌）显著高于趾长伸肌（快肌）。而Sox6的表达水平则相反。分析发现，猪Sox6 3'UTR中含有8个与miR-499-5p结合的位点。小鼠上，过表达miR-499-5p可使快肌纤维转化为慢肌纤维。基于"miR-499-5p通过抑制猪Sox6等靶基因表达调控猪肌纤维类型转化"的科学假设，本项目拟通过体内外实验弄清miR-499-5p是否可调控猪肌纤维类型的转化，鉴定猪Sox6为其靶基因，采用iTRAQ定量蛋白质组学分析筛选更多潜在的靶基因并验证其正确性，通过体内外实验确定miR-499-5p诱导的猪Sox6等靶基因表达抑制是否在miR-499-5p调控猪肌纤维类型转化中发挥了重要的作用。开展本项目研究，不仅可揭示猪肌纤维类型转化的调控机制，也可为改善猪肉品质提供重要的理论依据和新的思路。

肌纤维类型与猪肉品质密切相关。本项目成功分离获得了高纯度猪骨骼肌卫星细胞，成功克隆了猪Sox6完整编码区和3'非翻译区，证实了猪Sox6为miR-499-5p的靶基因。体内外研究结果表明，猪Sox6可促进肌纤维类型由慢肌转化为快肌。功能研究结果表明，miR-499-5p靶向Sox6促进猪慢肌纤维基因表达。除猪Sox6外，我们还对通过iTRAQ定量蛋白质组学筛选和文献获得的另外两个miR-499-5p靶基因（猪Histone H2A.Z和Thrap1）进行了研究。结果表明，miR-499-5p可抑制猪骨骼肌卫星细胞中Histone H2A.Z和Thrap1的表达。RNA干扰抑制猪Histone H2A.Z或Thrap1表达均可显著提高MyHC I和MyHC IIa的蛋白表达水平。研究同时发现，RNA干扰下调猪Histone H2A.Z 和Thrap1表达的作用均可被miR-499-5p inhibitor所解除，miR-499-5p inhibitor抑制MyHC I和MyHC IIa表达及促进MyHC IIb表达的作用也均可被猪Histone H2A.Z siRNA或猪Thrap1 siRNA所缓解。此外，本项目还研究了猪Sox6蛋白主动免疫对猪肉品质及肌球蛋白重链亚型表达的影响，结果表明，4 mg/头猪Sox6蛋白主动免疫生长育肥猪可显著提高肌肉红度值和大理石纹含量并显著上调肌肉中MyHC I的表达。综上结果表明，miR-499-5p 通过Sox6、Histone H2A.Z或Thrap1调控猪肌纤维类型转化，猪Sox6蛋白主动免疫生长育肥猪可通过改变肌纤维类型组成进而改善肉品质。项目研究结果不仅揭示了猪肌纤维类型转化的调控机制，也为改善猪肉品质提供了重要的理论依据和新的思路。