miR-214介导Hedgehog信号通路调控鳜幼鱼肌纤维增殖机理研究

骨骼肌既是鱼类的主要结构组织，也是维持机体代谢稳态和免疫应答的重要场所，其正常发育对鱼类的生长、健康及品质至关重要。我们前期研究发现miR-214在鳜幼鱼肌肉的表达显著高于成鱼，而抑制miR-214导致肌纤维增殖水平、Hedgehog (Hh)信号活性和MyoD表达降低，我们推测三者可能协同调控鳜肌纤维增殖。本项目拟进一步在体内外干扰miR-214表达和Hh信号活性的条件下，采用冰冻切片和免疫荧光技术检测肌纤维增殖水平的变化；利用荧光素酶报告技术确证Sufu是miR-214调控Hh信号通路的靶基因；继而采用整胚原位杂交、荧光素酶报告技术和胚胎显微注射过表达载体等实验确定Hh信号通路通过激活MyoD表达促进肌纤维增殖，以阐明miR-214介导Hh信号通路调控鳜肌纤维增殖的分子机理。本研究以期揭示鱼类肌纤维增殖调控的分子机理，并为培育具有肌纤维快速增殖能力的特种鳜鱼新品种提供理论支撑。

Skeletal muscle is not only the main structure, but also an important tissue to maintain metabolic homeostasis and immune response in fish, its normal development is crucial for fish growth, health and quality. In our previous study, miR-214 showed significantly higher level of expression in muscle of Chinese perch juveniles than in adults, and inhibition of miR-214 resulted in decreased myofiber hyperplasia, Hedgehog (Hh) signaling pathway activity and MyoD expression. Therefore, we speculate that miR-214, Hh signaling pathway and MyoD may have synergistic effects on the regulation of myofiber hyperplasia. This project intends to further reveal the effect of interfering with expression of miR-214 and activity of Hh signaling (in vivo and in vitro) on myofiber hyperplasia by using frozen section and immunofluorescence technology. Then we will use luciferase reporter system to verify if miR-214 could regulate Hh signaling pathway by inhibiting Sufu. Further, whole-mount in situ hybridization, luciferase reporter system and embryo microinjection of overexpression vector will be carried out to determine that Hh signaling pathway is mediated by miR-214 and then induced MyoD expression to promote the myofiber hyperplasia of Chinese perch. Finally, the molecular mechanism of miR-214 mediated Hh signaling pathway that promoting the myofiber hyperplasia of Chinese perch juvenile will be elucidated. It is expected that the obtained results will make significant contribution to reveal the molecular mechanism of fish myofiber hyperplasia. More importantly, these findings will also provide theoretical supports for the cultivation of new species of Chinese perch with the ability of rapid hyperplasia of myofiber.