小型猪IGF-1基因变异在发育性骨生长中的作用及机制

Studies have implicated the key regulatory roles of IGF-1 for miniature body size of mini-pig via direct or indirect (regulation of skeletal muscle) control of bone developmental growth. Although the associations between IGF-1 SNPs and pig body size were found in our previous studies, molecular studies exploring the function and mechanism for the association is on need urgently. In this research, in order to investigate the consequences of IGF-1 polymorphisms on the gene expression, structure and function, the functional significance of this gene polymorphisms will be analyzed including the influence of CA STR at the 5'-end on the promoter activity; the explicit effect and mechanism of the exonic mutations on the IGF-1 expression, structure and regulation of bone developmental growth; the interactions of the different 3'-UTR with miRNAs thus the regulation to the IGF-1 expression; the association and the regulated mechanism of the three E-peptides and the reverse overlapped LncRNA on the gene expression and the impacts to the skeletal muscle. These mechanistic studies are not only the continuation of previous work, which may be particularly important when it comes to SNPs associations with IGF-1 expression and function by the new regulated mechanism of the variable responses in miniarture and large pig breeds. The study provides theoretical premise for the research and development of miniature pigs.

IGF-1作为直接或通过作用于骨骼肌对动物发育性骨生长起决定性作用的因子在猪体型形成中起关键作用，本课题组前期解析了小型猪IGF-1全基因序列的基因结构、SNPs及与体型性状的相关关系，但其对IGF-1的影响及分子机制有待阐明。本项目拟深入阐明小型猪IGF-1基因启动子区CA STR多态性对其启动子活性及与骨生长发育相关通路的影响，外显子变异对IGF-1表达、结构及调控发育性骨生长的作用及机制，差异3'UTR与miRNA作用的异同及对IGF-1表达的调控，不同E肽可变剪接体和反向重叠基因LncRNA在不同体型性状猪中与IGF-1表达和其对骨骼肌作用的关系及调控机制，全面解析小型猪IGF-1基因各个潜在有意义的SNP对其表达、结构及对发育性骨和骨骼肌生长的影响及机制，提出SNP影响猪IGF-1表达及功能的新的分子机制，其结果可以为小型猪研究和开发利用提供理论前提。

IGF-1通过直接作用于骨组织或作用于骨骼肌来间接调控发育性骨生长，从而在猪体型形成中起关键作用。本课题组前期解析了小型猪IGF-1全基因序列的基因结构、SNPs及与体型性状的相关关系，但由其关键SNPs及独特的基因结构差异引起的IGF-1的变化对发育性骨生长的影响及分子机制有待阐明。本项目分别对小型猪IGF-1基因启动子区、外显子区和3'UTR的SNPs对骨生长发育的影响进行深入解析，并针对IGF-1可变剪接体及关键lncRNA对小型猪的体型形成机制进行探讨。结果表明：1. 猪IGF-1基因启动子区的不同长度的CA微卫星重复序列通过与HIF1α转录因子结合差异在调节IGF-1表达过程表现出不同的转录活性；2. 猪IGF-1基因C区编码基因存在的同义突变c.258A>G影响IGF-1基因在转录水平和翻译水平的表达量及稳定性，并且导致不同基因型编码产生的IGF-1与受体IGF-1R结合差异；3. IGF-1的表达受其3'UTR的SNPs和miR-new14的影响，miR-new14通过调控AKT和ERK信号通路抑制细胞增殖，促进细胞凋亡，并且IGF-1参与到这些通路中；4. T-MGF19E 肽能显著促进MC3T3-E1细胞的增殖、分化和矿化，并具有促进骨损伤愈合的作用；5. 猪lncRNA-MEG3中筛选出8个SNPs，其中4个SNPs（g.3087C >T, g.3108C >T, g.3398C >T和g.3971A >C）与肉品质显著相关，由其形成的两种单倍型的过表达不同程度地激活了JAK2/STAT3信号通路，促进了猪骨骼肌卫星细胞（SCs）的分化。以上结果全面解析了小型猪IGF-1基因各关键SNPs及独特的基因结构差异（E肽和lncRNA-MEG3）对其表达、结构及对发育性骨和骨骼肌生长的影响及机制，为小型猪的开发利用提供理论前提。