GRP78 AS lncRNA调控猪骨骼肌纤维脂质沉积的作用机制研究

To improve pork quality and overcome the supply crisis of domestic high quality pork, it is greatly significant to systematically explore lipid deposition of pig skeletal muscle fiber. Endoplasmic reticulum (ER) is the key place of cellular lipid synthesis, and ER stress which is regulated by GRP78 (glucose-regulated protein 78 kDa) significantly affects regulation of lipid deposition. Studies showed that there were close correlations among lncRNA (long non-coding RNA), lipid deposition of pig skeletal muscle fiber and ER stress, but the regulatory mechanism is still unknown. Moreover, the results of other species lncRNA may be unsuitable reference for porcine lncRNA because of their poor conservation in different species. Therefore, based on our previous sequencing analysis of lncRNAs on skeletal muscle cells and intramuscular adipocytes between obese-type Bamei pig and lean-type Large White pig, we found that a novel porcine lncRNA——GRP78 AS (antisense) lncRNA not noly potentially sponged miR-130a which targeted the key gene of fat deposition——PPARg (peroxisome proliferator-activated receptor g), but also potentially bound to GRP78 mRNA in partial complementary pairing, which was the vital regulatory gene of ER stress, forming an mRNA/AS lncRNA duplex. However, the effect and regulatory mechanism of GRP78 AS lncRNA on lipid deposition in pig skeletal muscle fiber is still unknown. In this study, we will explore the effect and molecular mechanism of GRP78 AS lncRNA on lipid deposition of pig skeletal muscle fiber through PPARg and GRP78 signal pathway using RNA-FISH (fluorescence in situ hybridization), RPA (RNase protection assay), knockout, knockin, RNA antisense affinity selection and mass spectrometry, RNA-IP (RNA-Immunoprecipitation) and other technologies. Moerover, we will illuminate the genetic correlation between GRP78 AS lncRNA gene and lipid deposition trait of skeletal muscle fiber in obese-type and lean-type pigs. The results of this project will provide novel candidate lncRNA gene for the selection and breeding of pigs with ideal lipid deposition trait of skeletal muscle fiber to improve pork quality.

猪骨骼肌纤维脂质沉积调控机制研究对改善肉用性能，缓解国内优质猪肉供应危机具有重大意义。内质网是细胞内脂质合成的关键场所，内质网应激对脂质沉积具有重要调控作用。研究表明，lncRNA与骨骼肌纤维脂质沉积和内质网应激密切相关，但其机制不清楚，物种间保守性差，致使其研究结果参考价值有限。因此，申请人基于脂肪型八眉猪和瘦肉型大白猪骨骼肌细胞与肌内脂肪细胞lncRNA测序和生物信息学分析，发现猪GRP78 AS lncRNA不仅潜在吸附靶向脂质沉积重要基因PPARg的miR-130a，而且与内质网应激关键基因GRP78 mRNA有互补结合区域，但其作用和机制不清楚。基于此，本项目将利用FISH、RPA、IP、敲除和敲入等技术探索该lncRNA通过PPARg和内质网应激通路调控猪骨骼肌纤维脂质沉积的作用机制，解析该lncRNA基因与两种类型猪骨骼肌纤维脂质沉积的遗传相关，为改善猪肉用价值提供科学依据。

猪骨骼肌纤维脂质沉积调控机制研究对改善肉品质，缓解国内优质猪肉供应危机具有重大意义。为了探究骨骼肌纤维脂质沉积调控机制，本项目通过转录组学和生物信息学的联合分析，鉴定了一条与细胞生长发育、脂质沉积都相关的lncRNA，命名为GRP78 AS lncRNA。研究表明，敲低GRP78 AS lncRNA在猪肌卫星细胞中抑制成肌标志基因的表达，抑制猪肌卫星细胞分化成肌管，并促进成脂标志基因的表达进而促进肌管内脂质沉积。同时，GRP78 AS lncRNA与PPARG竞争结合miR-130a-3p，双荧光素报告酶试验显示miR-130a-3p与PPARG直接结合，通过转录后抑制PPARG的mRNA表达，从而抑制成肌细胞脂质沉积。GRP78 AS lncRNA还与内质网应激相关，通过调控内质网应激相关基因影响成脂。此外，项目组还从营养水平、关键基因和ncRNA多方面研究了骨骼肌纤维脂质沉积。研究发现，异亮氨酸可以促进肌细胞脂质沉积，并使其线粒体发生空泡化，线粒体功能受到抑制；功能基因GABPα对猪肌卫星细胞、小鼠成肌细胞都具有促进脂质沉积的作用；功能基因DNMT3A可通过促进PPARG的甲基化降低其表达水平，进而抑制脂质沉积。另外，鉴定了lnc-ORA、lncIMF4、lncIMF2等长链非编码RNA以及miR-146a-5p与miR-332-3p等miRNA作为调控骨骼肌纤维脂质沉积或肌内脂肪细胞脂质沉积的潜在靶标，发现lnc-ORA和miR-332-3p对脂肪组织、肌肉组织均具有功能；lncIMF4、lncIMF2和miR-146a-5p影响猪肌内脂肪细胞的增殖与成脂分化。因此，本项目研究结果完善了非编码RNA对猪骨骼肌纤维脂肪沉积的调控，为探索我国地方猪品种肉质优良特性的非编码RNA调控提供了新的候选基因，也为优质瘦肉型猪品种的选育和健康优质猪肉生产提供科学依据。此外，本项目还从营养水平，功能基因和非编码RNA的多角度对猪的脂肪沉积和肌肉发育进行了研究，这些成果为从遗传和营养角度改善猪肉品质提供了新的思路和理论依据。