猪MSTN通过MEF2C/miR222/SCD5特有分子通路调控脂肪酸代谢的机理研究

Functional loss of MSTN not only results in the “double muscling” phenotype in animal, but also a significant decrease in body fat. The molecular mechanism behind it, however, remains to be elucidated. Taking advantage of the MSTN knockout cloned pigs created by our group, we performed the mRNA and miRNA transcriptomic sequencing using the subcutaneous fat tissues and found that, with the knockout of MSTN, MEF2C and miR222 were up-regulated, while SCD5 was down-regulated. Bioinformatic analysis predicts that MEF2C has a binding motif in the promoter of miR222, and miR222 has a putative binding site in the 3´ UTR of SCD5. Based on these observation the applicant hypothesizes that MSTN is likely to regulate fatty acid metabolism through cascade signaling with MEF2C/miR222/SCD5. In order to address this hypothesis, the project, at the molecular and cellular level using Chip, EMSA and RIP-PCR, etc, would investigate the transcriptional activation of miR222, as well as the translational inhibition of miR222 upon SCD5 concurrent with the functional ablation of MSTN. At the metabolic level, it would elucidate the effect of MSTN on fatty acid metabolism coupled with the MEF2C/miR222/SCD5 cascade signaling. Next，the regulatory role of MSTN upon the three downstream genes would be validated by a rescue cell model. Finally, the reliability of MSTN’s effect on fatty acid metabolism through MEF2C/miR222/SCD5 signaling would be proved in the MSTN bi-allelic knockout pigs. The experimental result would discover the molecular mechanism of MSTN to regulate fatty acid metabolism and identify a novel molecular pathway for fat deposition. It also would give rise to potential inviting targets for pork quality improvement.

MSTN失活使动物产生“双肌”表型，也引起体脂显著减少，但其调控脂肪沉积的分子机理不甚明了。借助前期研制的MSTN基因敲除猪，通过皮下脂肪转录组测序，发现MSTN失活引起MEF2C和miR222上调、SCD5下调，且预测到MEF2C在miR222启动子上有结合基序，而miR222在SCD5的3´UTR有潜在靶点。据此，申请人提出如下假设：MSTN失活→上调MEF2C→上调miR222→靶向沉默SCD5，四者形成分子通路，抑制饱和脂肪酸脱氢。本项目拟在分子和细胞水平，探明在MSTN缺失时，MEF2C、miR222与SCD5的级联调控关系；在代谢水平，通过分析脂肪酸合成与含量变化，明确MSTN经由上述通路调控脂肪酸代谢；再利用回复表达与纯合突变个体，反证上述通路与脂肪酸代谢的关系。研究结果将揭示MSTN调控脂肪酸代谢的分子机理，挖掘出猪脂肪沉积的特有调控通路，为改良猪肉品质提供新靶点。

MSTN失活使动物产生“双肌”表型，也引起体脂显著减少，但其调控脂肪沉积的分子机理不甚明了。借助前期研制的MSTN基因敲除猪，通过皮下脂肪转录组测序，发现MSTN失活引起MEF2C和miR222上调、SCD5下调，且预测到MEF2C在miR222启动子上有结合基序，而miR222在SCD5的3´UTR有潜在靶点。据此，本研究提出如下假设：MSTN失活→上调MEF2C→上调miR222→靶向沉默SCD5，四者形成分子通路，抑制饱和脂肪酸脱氢。本项目的研究目标是探明猪MSTN通过MEF2C/miR222/SCD5特有分子通路调控脂肪酸代谢的机理。开展的主要研究内容包括：（1）MSTN影响猪脂肪酸代谢和基因表达的研究；（2）MSTN通过转录因子MEF2C调控miR222表达的机制研究；（3）MSTN通过MEF2C/miR222途径影响SCD5表达的机制研究；（4）MSTN回复表达验证其对MEF2C/miR222/SCD5的调控效应；（5）MSTN敲除纯合猪的获得与分子通路功能验证。取得的重要结果与关键数据有：（1）MSTN失活导致背膘厚度降低、肌内脂肪含量减少同时甘油三酯含量降低了58%。（2）MSTN失活后MEF2C表达量上升约2.3倍，同时MEF2C靶向miRNA222启动子区域促进miRNA222表达量为原来的2倍。同时ChIP和EMSA试验表明，miRNA222启动子区域存在MEF2C的结合位点。（3）qPCR、WB结果显示，MSTN失活后SCD5表达量下降了60%，气质联用检测脂肪酸组成结果显示，MSTN失活后C18含量显著下降，而加入miRNA222模拟物时，C18含量显著上升。（4）回复表达MSTN结果显示，MEF2C表达量显著下降P<0.01，miRNA222和SCD5的表达量无显著变化。（5）qPCR、WB结果显示，杂合敲除的猪（MSTN+/-）与纯合敲除的猪（MSTN-/-）相比，MSTN表达量显著降低，MEF2C表达量显著上升。该项目的科学意义有：揭示了MSTN调控脂肪酸代谢的分子机理，挖掘出猪脂肪沉积的特有调控通路，为改良猪肉品质提供新靶点。