胰岛素样生长因子-1受体对巴马香猪体型矮小的调控机制研究

As a dwarf breed, Bama Mini-pigs is widely applied into the biomedical researches. At present, the formation mechanism of “proportional dwarfism” of the mini-pigs is poorly understood. Studies indicated that IGF-1/IGF-1R deficiency is critical in animal dwarfism. In our previous studies, unique haplotypes were found in the coding region, promoter region and 3’UTR of the IGF-1R gene in Bama Mini-pigs. In this study, we will first elucidate the effect of the different haplotypes located in the coding region of IGF-1R gene on the rate and conformation of the protein folding, clarify the influence on the phosphorylation level of the membrane protein and the transmission of the relevant signal pathway and expound the conformation change on the receptor-ligand interaction, then further compare the difference of cell proliferation, differentiation and migration among different haplotypes; secondly, we will analyze the expression difference of the IGF-1R caused by the SNPs located in the non-coding region through the levels of transcription and translation; thirdly, the existence and distribution of the alternative splicing from different pig breeds will be explored the significance; In addition, the relations between the overlapping gene and IGF-1R gene will be analyzed. In conclusion, this study will be a comprehensive in-depth analysis of the influence of SNPs on the function of IGF-1R in mini-pigs and its molecular mechanism, which may be particularly important when it comes to SNPs associations with IGF-1R expression and function by the new regulated mechanism of the variable responses in mini-pigs and large pigs. Furthermore, this study will lay foundations to the Bama mini-pigs as model animals on growth and development.

巴马香猪作为矮小品系应用于生物医学研究，目前对其比例性矮小机制尚不明确。研究表明IGF-1和IGF-1R缺乏在其体型矮小中起关键作用。前期研究发现巴马香猪在IGF-1R基因编码区、启动子、内含子及3’UTR存在独特单倍型，本项目拟检测IGF-1R编码区不同单倍型对蛋白质折叠速率、构象的影响，配体-受体间相互作用、自身磷酸化水平及相关信号通路的差异及不同单倍型对细胞增殖、分化和迁移的影响；从基因转录、翻译水平明晰其非编码区SNPs对IGF-1R表达量的改变；观察不同猪种IGF-1R可变剪切体存在及分布，阐明其功能及对IGF-1R主转录本的干扰；明确IGF-1R启动子区的重叠基因与IGF-1R表达之间的线性关系。综上，本研究将全面深入解析SNPs对小型猪IGF-1R功能的影响及其分子机制，提出猪IGF-1R表达及功能新的调控机制，为巴马香猪作为研究机体生长的模式动物奠定理论数据。

阐明IGF-1R基因变异的作用能够为探索小型猪体型矮小的形成机制提供理论支持，本项目以大白猪和巴马香猪作为研究对象，拟阐明巴马香猪在IGF-1R基因启动子、编码区及3’UTR存在的单倍型及其功能变异，观察不同猪种IGF-1R可变剪切体存在及分布特征，得到如下结果：（1）发现猪IGF-1R基因核心启动子位于 -1~-1555 bp区域内。不同品种猪IGF-1R基因启动子区-1 468 bp处存在G/C突变、-801 bp处存在A/C突变及-561 bp处存在一个GCGGCG插入，这些突变和插入可能造成不同品种猪IGF-1R表达量的差异。（2）IGF-1R胞外域编码区的两种单倍型影响基因的转录、翻译、蛋白质构象以及其与IGF-1的结合情况，进而影响IGF-1R下游通路的激活，最终导致成骨细胞分化的差异（3）IGF-1R胞内域编码区的5个同义突变影响基因的转录、翻译、自身磷酸化，进而影响IGF-1R下游p-AKT/p-ERK的水平，最终导致PK-15和C2C12细胞增殖的差异。（4）猪IGF-1R基因的可变剪接体及其表达谱尚未阐明。本研究在猪不同组织中鉴定出IGF-1R基因的主转录本ISO01及一条新的可变剪接体AS02，检测到AS02在软骨组织中的表达水平显著高于肌肉和肝脏组织，而ISO01却呈现相反的趋势，表明IGF-1R可变剪体接的表达量可能影响主转录本的表达。（5）本研究发现巴马香猪与长白猪中差异表达的miR-322、miR-140和miRNA Y-56均靶向IGF-1R 3'UTR并抑制其表达。miR-322通过靶向IGF-1R和INSR的3'UTR抑制其表达水平并加重地塞米松诱导的肌肉萎缩；miR-140通过靶向IGF-1R和SOX4的3'UTR直接或间接地抑制IGF-1R的表达量，从而抑制PFFs的增殖；miRNA Y-56通过靶向IGF-1R 3'UTR下调其表达水平，进而抑制PSCs的增殖和周期进程。以上研究为小型猪的体型矮小形成机制提供了理论数据。