新发现的LncRNA TCONS_00323213基因的表达规律及其对猪骨骼肌卫星细胞分化与骨骼肌发育的表观遗传调控

The expression patterns of LncRNA TCONS_00323213 in the different tissues and skeletal muscle satellite cells at the differently differentiated time points in pigs was firstly found and identified by us. However, the molecular feature of this lncRNA, its regulation pattern for target genes and the Epigenetic mechanisms for regulating the differentiation of pig skeletal muscle satellite cells and the development of skeletal muscle were not understood at present. Based on the previous study results, we are going to clone and identify the molecular characteristics of LncRNA TCONS_00323213 gene by using Bioinformatics and Northern Blot techniques, detect the transcriptional activity and the binding of the transcription factors by Dual-luciferase reporter assays, site mutation technique and Chromatin Immunoprecipitation (ChIP), identify the expression patterns and functional clusters of the target genes by RNA interference (RNAi) and Overexpression, High-throughput sequencing, and fluorescence-activated cell sorting (FACS) technique, analyze the functional interaction of lncRNA with the target proteins by RNA-pulldown, SDS-PAGE and mass spectrometry techniques and then validate them by deletion-mapping and RNA immunoprecipitation (RIP) techniques, quantitatively detect the mutual effects of the target genes regulated with this lncRNA and the interaction proteins by utilizing RNAi, ChIP-seq and ChIP-qPCR techniques, and obtain the transgenic pig population and the phenotypes related the skeletal muscle development by CRISPR-Cas9 and Immunohistochemical techniques (IHCT). Collectively, the results will reveal the molecular characteristics of LncRNA TCONS_00323213 gene, uncover the regulation patterns of its target genes and the Epigenetic mechanisms of the differentiation of skeletal muscle satellite cells and the development of skeletal muscle in pig and provide the theoretic basics and technique supports for finding novel gene resources with national intellectual property.

我们首次鉴定了猪LncRNA TCONS_00323213基因在组织与骨骼肌细胞分化中的时空表达规律，但其分子特征、表达调控模式以及调控骨骼肌卫星细胞分化与骨骼肌发育的分子机制几乎一无所知。有鉴于此，本课题拟在前期研究的基础上，利用Northern Blot等技术鉴定该lncRNA分子特征；利用定点突变及ChIP技术确证其启动子区活性及转录因子的结合；应用RNAi/Overexpression、ChIP-Seq、RNA-pulldown、质谱技术等鉴定其与目标蛋白的互作及调控靶基因表达模式；应用CRISPR-Cas9和IHCT技术获得基因编辑猪群体和不同日龄骨骼肌发育表型变化数据；在分子、细胞、个体和群体水平上阐明该lncRNA调控猪骨骼肌卫星细胞分化与骨骼肌发育的分子机制，为改良猪骨骼肌生长发育性状提供具有我国自主知识产权的基因素材和技术依托。

肌源性分化是一个复杂而精密的生物过程，受多种因素的调控，其中lncRNA起着非常重要的作用，但对其调控机制的深入研究却很有限。本研究发现，一个新的lncRNA TCONS_00323213影响猪卫星细胞（PSCs）增殖和分化。本研究通过定量聚合酶链反应（qPCR）、RACE、RNA-seq、5-乙炔基-20-脱氧尿苷（EdU）、Western Blot（WB）、免疫荧光染色（IF）、pull down检测、质谱分析（MS）、RNA免疫沉淀检测（RIP）、CUT & Tag检测进行研究，鉴定了lncRNA TCONS_00323213的分子特征与表达规律，lncRNA TCONS_00323213 敲低后的RNA-seq结果表明lncRNA TCONS_00323213通过参与多种信号通路参与肌肉发育。功能分析表明，lncRNA TCONS_00323213抑制了PSCs的增殖，这一点从EdU阳性细胞的百分比和PSCs增殖期间的细胞存活率可以看出。lncRNA TCONS_00323213在成肌过程中促进了细胞分化，增强了肌原蛋白（MYOG）、MEF2C和MYHC。Pull down和RIP实验表明，lncRNA TCONS_00323 213与PKNOX2相互作用，而PKNOX2在PSCs分化中起着转录调节的作用。CUT & Tag实验表明，在lncRNA TCONS_00323213敲除后，PKNOX2在MYOG启动子上有明显的富集。我们的研究结果初步确立了lncRNA TCONS_00323213与PKNOX2相互作用，解除PKNOX2对MYOG启动子的抑制作用，并增强MYOG的表达以促进PSCs分化的新联系。这种新型的lncRNA TCONS_00323213的作用可能为理解lncRNA在猪肌体发育中的分子调控提供新的启示。在本项目的资助下，发表SCI论文5篇，发明专利3项。