印记基因H19的表观调控及其在骨骼肌中的作用

Our previous study showed that miR-675 and H19 had a trend of rising expression while the differentiation time of skeletal muscle cell line increased. But the expression level of the former was significantly higher than the latter. Both of them were highly expressed in the skeletal muscle tissue but lowly or not expressed in the other tissues. The mechanisms for that are not very clear at present. Therefore, our scientific hypotheses based on previous results are: the expression of miR-675 is possibly suppressed in the cell line and tissues that will promote the expression of H19. At the same time, the methylation change of core promoter of H19 will have effect on the binding of the transcript factors and increase the expression of H19, accordingly. At last, they have an important role in the differentiation of skeletal muscle and muscle development in pig. We plan to detect the expression of H19 and observe the change of cell morphology to determine the regulating effects of the miR-675 on H19, through the suppression expression and over-expression of miR-675. Otherwise, we isolate and identify the transcription factors (TF) binding to the H19 promoter to clarify the effects of the methylation on the TF binding and H19 expression and definite confirm the role of that in the differentiation of skeletal muscle cell line and muscle development in pig by DNA methylation sequencing and chromatin immunoprecipitation (ChIP). The epigenetic regulation mechanism of the effects of DNA methylation, miR-675 and H19 expression on the differentiation of skeletal muscle cell line and muscle development will be revealed and the novel genes and molecular markers related skeletal muscle development will be developed and utilized in pig production.

我们前期的研究表明，miR-675和H19在骨骼肌细胞中随着分化时间的增加呈现上升表达趋势，且前者的表达量远远低于后者；在组织中，二者在肌肉中却都高表达，在其他大部分组织不表达或者很低表达。但其作用机理至今不甚明了。据此我们的科学假设是：miR-675在细胞和组织中的表达可能被抑制了，其具有促进H19表达的作用，同时，启动子甲基化改变可能影响了转录因子的结合，进而促进了H19的表达，影响了骨骼肌细胞分化和肌肉发育。通过miR-675抑制表达和超表达检测H19的表达，确定miR-675对H19的调控作用。通过甲基化测序和ChIP等实验鉴定H19的转录因子以及阐明甲基化对转录因子结合进而对H19表达的作用，确定其对骨骼肌细胞分化和肌肉发育的影响。最终明确甲基化和miR-675对H19表达的表观遗传学调控及其对肌肉发育的作用机制，为肌肉发育相关基因或分子标记的发掘提供新素材。

猪H19/IGF2印记基因的表达调控模式以及与miR-675等基因的互作及其调控骨骼肌发育的机理目前未见报道。本文通过猪骨骼肌定量表达、启动子活性检测和甲基化分析等实验完成了H19、IGF2、互作基因在猪骨骼肌及C2C12细胞分化中的表达规律，并对C2C12成肌分化过程中H19启动子甲基化水平、转录调控规律进行了研究。取得了如下结果：1.猪胚胎50d骨骼肌中H19、IGF2的表达水平略低于95d，但胚胎期表达水平都极显著高于成年期；而梅山猪在这50d和95d骨骼肌中的表达显著高于大白猪，但成年期恰好相反；2.Let-7家族基因在梅山猪和大白猪上述三个时间点表达模式具有显著差异，其表达水平与背最长肌pH值显著相关；3. H19、IGF2、INS2在C2C12分化过程中的表达模式都是先上调后下降，但H19上游0-4K序列是没有启动子活性的；4.发现H19上游0-4K在C2C12细胞分化0d和4d共有34个差异甲基化位点，主要集中在H19的第四个CTCF结合位点附近，且整体甲基化水平4d显著高于0d，而H19表达趋势相反。这些研究为H19/IGF2印记基因及其互作miRNA或转录因子在猪骨骼肌发育中的表观遗传作用机制提供了直接证据。. 发表基金标注SCI论文1篇（Li et al. BMC Genomics 2014, 15:811），授权专利1项，获得湖北省杰出青年基金资助1人，培养博士生和硕士生3人。国际动植物基因组亚洲会议（PAG Asia）和国际动物遗传学大会（ISAG）分组报告和墙报展示各1份。

内容获取失败，请点击重试