Hnrnpk基因在猪成肌细胞增殖分化过程中的表达及分子调控机制研究

Hnrnpk （heterogeneous nuclear ribonucleoprotein K）is a multifunctional protein that participate in chromatin remodeling, transcription, RNA splicing, mRNA stability and translation. Previous studies showed that Hnrnpk may play an important role in skeletal muscle cells proliferation and differentiation, but how and why it has this effect remains unknown. Our former research found that Hnrnpk was differentially expressed in the porcine skeletal muscle of different development stages by proteome analysis. Based on this observation, the subject was propounded. To reveal molecular egulatory mechanisms and signal pathway of Hnrnpk gene in porcine skeletal myoblasts proliferation and differentiation, several technologys were used, such as overexpression, RNAi, cell transfection, immunoblotting, immunohistochemistry, immunofluorescent, qRT-PCR, flow cytometry and transmission electron microscope in this study. Experimental investigation mainly contained: Experimental investigation mainly contained: the regulation effect of Hnrnpk in the expression of transcription c-Myc and activity of tyrosine kinase c-Src; the subcellular localization and expression of phosphorylated Hnrnpk in porcine myoblasts; the effects of ERK and c-Src inhibitor for Hnrnpk. The project lays a foundation for illustrating the regulatory mechanism of the skeletal muscle development and regeneration, and is helpful for increasing the meat production and meat quality of livestock/poultry genetic improvement and controlling the muscle regeneration of human being.

Hnrnpk是一个多功能蛋白，参与DNA修复、转录和翻译调控、RNA的剪切加工等生物学过程。以往研究表明，Hnrnpk可能通过多种途径对骨骼肌细胞增殖与分化起重要调控作用，但其作用机制及信号通路尚不清楚。我们前期蛋白质组学研究发现Hnrnpk在梅山猪不同发育时期骨骼肌中差异表达。基于此，本项目拟结合过表达、RNA干涉、细胞转染、免疫印记、免疫组化、免疫荧光、qRT-PCR、流式细胞、透射电镜等生物学手段，以猪成肌细胞为体外模型，以Hnrnpk调控转录因子c-Myc和酪氨酸磷酸化激酶c-Src的活性、ERK和c-Src阻断剂对Hnrnpk的影响及磷酸化Hnrnpk的亚细胞定位和作用为切入点，研究Hnrnpk基因对猪成肌细胞增殖分化的调控作用及信号通路。项目的研究结果不仅能为阐明骨骼肌发育和再生的复杂网络调节机制奠定基础，而且还可望为畜牧业提高肉产量和肉品质以及医学上控制肌肉再生提供依据。

完成了计划内容，获得猪Hnrnpk基因cDNA全长，通过RACE分析发现存在8种不同的转录本，由外显子1、8及17的选择性剪接引起，且8种不同的转录本对应4种蛋白；猪Hnrnpk基因时空表达分析显示其在骨骼肌和脂肪组织中高表达，猪骨骼肌发育过程中呈下调表达；亚细胞定位表明Hnrnpk主要位于细胞核内，胞浆中有少量表达，N端NLS和 C端KNS信号对其定位有重要影响，免疫荧光结果显示Hnrnpk在C2C12分化过程中有明显的出核表达；通过超表达和RNAi Hnrnpk基因以及ERK抑制剂PD98059和c-Src抑制剂PP2 处理揭示Hnrnpk对C2C12成肌细胞起着促增殖抑制分化的作用；Co-IP、GST-Pull Down和酵母双杂交以及RIP分析揭示Hnrnpk与c-Src在蛋白质和mRNA层次存在互作并调控其表达和功能。基于上述结果，提出了在成肌细胞增殖分化过程中Hnrnpk通过调控c-Src来发挥作用的调控模型。对猪c-Src基因进行一系列研究，包括克隆cDNA全长、时空表达分析、核心启动子鉴定等。到目前为止，已发表5文章，其中SCI收录1篇，另有1篇接受，待刊出， 2篇已提交，2篇正在整理中；培养博士研究生1名，硕士研究生4名。