chi-miR-99b-3p调控山羊肌肉发育及其与羔羊断奶应激的关系

MicroRNAs (miRNAs) regulate myocyte proliferation and differentiation, and have been confirmed to play important roles in the muscle development. Our previous studies found that the cell proliferation and differentiation related miRNA chi-miR-99b-3p is specifically high expressed in the muscle tissue of 7 month old goats (when the goats are growing rapidly) and this miRNA regulates multiple muscle development-related target genes. Therefore, we hypothesized that chi-miR-99b-3p may be involved in the regulation of goat muscle development. In addition, we also found that chi-miR-99b-3p was significantly decreased in early weaned lambs, suggesting that it may be associated with hindered growth of lambs after weaning. In this study, the potential targets of chi-miR-99b-3p will be identified by dual fluorescence experiments. Subsequently, the spatiotemporal expression of chi-miR-99b-3p and its targets in goats will be investigated by qRT-PCR technology. Finally, miRNA loss and gain strategies combined with sequencing and bioinformatics analysis will be used to clarify the regulation of chi-miR-99b-3p in goat skeletal muscle cells and its effect on goat skeletal muscle development under stress condition. This study will preliminarily clarify the molecular regulatory mechanism of chi-miR-99b-3p in goat muscle development, which may provide a new theoretical basis for the breeding and efficient production of high quality meat goats.

MicroRNA（miRNA）参与肌细胞增殖和分化调控，在肌肉发育过程中起重要作用。我们前期工作发现，与细胞增殖分化相关的miRNA chi-miR-99b-3p在快速生长期的山羊骨骼肌中特异性高表达，且调控多个肌肉发育相关靶基因，揭示chi-miR-99b-3p可能参与调控山羊肌肉发育。此外，chi-miR-99b-3p在断奶羔羊中显著下调，推测其可能与断奶后羔羊生长受阻有关。为此，本研究拟首先鉴定chi-miR-99b-3p的靶基因，同时检测其在山羊中的时空表达规律，进一步综合利用miRNA过表达和干扰策略，结合测序和生物信息学技术系统研究chi-miR-99b-3p在山羊骨骼肌细胞中的调节作用及其在应激状况下对山羊骨骼肌发育的影响，初步揭示chi-miR-99b-3在山羊肌肉发育过程中的分子调控机制，为优质肉用山羊选育和高效生产提供新的理论依据。

miRNA在山羊肌肉发育过程中发挥着重要作用。chi-miR-99b-3p可能参与调控山羊肌肉发育，但具体的作用机制尚不清楚。因此，本项目主要开展以下研究：.运用miRNA靶基因预测软件，并根据相关的生物学通路分析，筛选出了chi-miR-99b-3p的潜在靶基因。运用双荧光素报告基因检测、qRT-PCR方法、Western blot等确定chi-miR-99b-3p的靶基因。综合利用miRNA过表达和干扰策略，结合测序和生物信息学研究chi-miR-99b-3p在山羊骨骼肌细胞中的调节作用。.过表达chi-miR-9b-3p后抑制了Caspase-3和NCOR3的表达水平，而在用chi-miR-99b-3p抑制剂转染后则促进了它们的表达，确定了chi-miR-99b-3p的靶基因Caspase-3和NCOR1。体外培养SMSC细胞，在SMSC分化过程中，内源性chi-miR-99b-3p表达增加，Caspase-3和NCOR1在SMSC分化的第3天和第5天的表达降低。.过表达chi-miR-99b-3p后，凋亡相关基因Caspase-9、7和BCL-2的表达和蛋白表达均下调，抑制chi-miR-99b-3p后则表达上调。过表达chi-miR-99b-3p显著增加细胞活力并抑制BCL2的蛋白表达量，与对照组相比，干扰chi-miR-99b-3p后，凋亡相关基因Caspase-9、Caspase-7和BCL2的表达显著增加。过表达chi-miR-99b-3p后细胞转录谱显示，34个chi-miR-99b-3p候选靶基因表达下调。miRNA测序发现chi-miR-99b-3p过表达诱导47个差异表达miRNA，其中16个上调，31个下调。KEGG分析表明，差异miRNA参与细胞周期、松弛素信号通路、DNA复制和蛋白质消化和吸收等通路，这与差异基因的结果一致。构建过表达chi-miR-99b-3p后细胞miRNA-mRNA相互作用网络，chi-miR-99b-3p可使ORC1、SLC6A17和TTC4表达下调。构建地塞米松应激细胞模型，过表达和抑制表达chi-miR-99b-3p后，结果和正常细胞的相似。.本研究揭示了chi-miR-99b-3p在骨骼肌卫星细胞增殖分化中的影响机制，为进一步研究其在骨骼肌生长发育方面的作用提供理论依据，丰富了山羊肌肉生长发育调控网络。