HOXA13调控猪肌纤维细胞中脂肪沉积的分子机理研究

Fat content is the key to meat quality improvement. The mechanism of fat deposition among muscle fibers, that is, intramuscular fat, was studied extensively. While the the mechanism of fat deposition in muscle fibers was still unclear. The previous study indicated that the fat content was significantly different in different types of skeletal muscle fiber. Our previous study indicated that HOXA13 was differently expressed in fast-twitch muscles and slow-twitch muscles. Over-expression of HOXA13 could promote muscle cells to differentiate into slow-twitch muscle fibers, and facilitate the lipid synthesis in muscle cells. How does HOXA13 exert its fat deposition promotion effect in muscle fibers? The previous study indicated that HOXA13 could inhibit the activity of TGF-β/Smad signaling pathway, while this pathway could inhibit lipid synthesis. Base on these results, we hypothesize that HOXA13 could facilitate lipid synthesis in muscle cells by inhibiting TGF-β/Smad signaling pathway. This project plans to overexpress or knockdown HOXA13, or use CRISPR/Cas9 to knockout HOXA13 in porcine skeletal muscle satellite cells, and then induce the muscle cells turn to lipid synthesis process. The expression of lipid metabolism related genes and the activity of TGF-β/Smad signaling pathway would be subsequently detected. This project also plans to use the activiting and blocking experiment to analyze the effect of TGF-β/Smad signaling pathway on mediating the regulation function of HOXA13 in lipid synthesis. We wish we could reveal the molecular mechanism of HOXA13 in regulating intramuscular fat deposition in skeletal muscle fibers, and provide the fundamental basis for pork quality improvement.

脂肪含量影响猪肉品质，肌纤维间脂肪（肌内脂肪）调控机制已有大量研究，但肌纤维内脂肪调控机制仍有待研究。研究表明不同类型肌纤维中脂肪含量显著差异。申请人前期发现HOXA13在快肌和慢肌差异表达，促进肌肉细胞向慢肌纤维分化、进而促进肌肉细胞中脂质合成。HOXA13通过何种途径促进肌纤维中脂肪沉积？研究表明HOXA13抑制TGF-β/Smad通路活性，而此通路抑制脂肪合成。申请人假设HOXA13通过抑制该通路促进肌肉中脂肪合成。本项目拟在猪骨骼肌卫星细胞超表达或干扰表达HOXA13、及用CRISPR/Cas9技术构建HOXA13敲除细胞，再诱导细胞合成脂肪，检测脂肪代谢关键基因和TGF-β/Smad通路活性变化；通过增强和阻断试验，分析TGF-β/Smad通路在介导HOXA13调控脂质代谢中的作用。本项目拟揭示HOXA13调控猪肌纤维中脂肪沉积的分子机理，为猪肉质性状改良提供理论基础和分子靶标。

肌内脂肪含量影响猪肉品质，并且不同类型肌纤维中脂肪含量显著差异。申请人前期确定了HOXA13在不同类型肌纤维中差异表达，具有促进肌肉细胞向氧化型肌纤维分化的能力，本项目继续研究HOXA13调控猪肌纤维中脂肪沉积的作用及其分子机理。本研究发现HOXA13在不同肌内脂肪含量的猪背最长肌中差异表达。利用HOXA13不同表达水平的肌肉细胞条件培养基（MCM）处理3T3-L1前体脂肪细胞，发现短时间MCM处理情况下，肌肉细胞中的HOXA13正向调节脂肪细胞的脂肪沉积；长时间MCM处理情况下，肌肉细胞中的HOXA13负向调节脂肪细胞的脂肪沉积。敲低HOXA13的MCM可能抑制脂肪细胞的脂肪酸合成和脂质分解过程，但促进其甘油三酯的合成。另外，敲低HOXA13后，肌细胞内脂肪沉积减少；将TGF-β/Smad通路的活性进行抑制，敲低HOXA13的效应没有得到挽救，说明TGF-β/Smad通路在HOXA13影响肌细胞脂质沉积过程中的作用有待进一步验证。本研究为肉质性状改良工作提供理论基础和分子靶标，对养猪业的发展和人们生活质量的提升具有重要意义。