MSTN/miR-124a-2/GR通路介导猪肌肉组织特异性调控肌内脂肪细胞分化的机制研究

揭示肌内脂肪沉积的组织特异性调控机制是选育提高猪肌内脂肪含量的关键。我们前期研究发现，肌肉组织中MSTN能够抑制肌内脂肪细胞糖皮质激素受体（GR）的水平，下调肌内脂肪细胞糖皮质激素敏感性从而抑制其分化，miR-124a-2参与了MSTN对脂肪细胞GR的抑制。本研究首先对猪MSTN/miR-124a-2/GR通路进行解析，并鉴定通路中关键的Smad结合位点（SBE）和miRNA结合靶点（miRBS）。进而采集二花脸猪SV细胞建立前体脂肪细胞系，采用CRISPR/Cas9技术构建SBE或miRBS敲除的突变前体脂肪细胞系，体外分析阻断MSTN/miR-124a-2/GR通路对脂肪细胞分化的作用。最后采用不同荧光标记突变和野生前体脂肪细胞，等量混合注射皮下脂肪和肌肉组织，通过脂肪细胞在体分化能力研究，探讨阻断MSTN/miR-124a-2/GR通路能否解除或减弱肌肉组织对肌内脂肪细胞的抑制作用。

Elucidation of tissue-specific regulation mechanims of fat deposition in muscle is crucial for improving intramuscular fat content. Our previous study found that muscle-derived MSTN inhibited glucocorticoid receptor (GR) level in intramuscular adipocytes, which led to decreased glucocorticoid sensitivity and reduced differentiation ability of intramuscular adipocytes. Our study also revealed that miR-124a-2 was involved in MSTN inhibition of GR. In this study, we will analyze the MSTN/miR-124a-2/GR pathway in pigs, identify the Smad binding element (SBE) and miRNA binding site (miRBS). Pig preadipocyte cell lines will be established by immortalizing SV cells derived from Erhualian pigs. Mutant preadipocyte cell lines with SBE or miRBS knockout will then be constructed by using CRISPR/Cas9. The effect of MSTN/miR-124a-2/GR pathway on preadipocyte differentiation will be analyzed in vitro using both mutant cell lines in which the pathway is blocked by SBE or miRBS mutations. Mutant preadipocytes will then be marked with green fluorescence and injected into subcutaneous fat as well as muscle tissues to allow differentiation in vivo. Wild preadipocytes marked with red fluorescence will be used as control. With in vivo experiment, we will verify whether the interruption of MSTN/miR-124a-2/GR pathway could attenuate the inhibition of muscle on intramuscular adipocytes differentiation.