miR-27在CLA降低草鱼肝脂积累中的作用及机理研究

Nutrition-associated liver disease (NALD) is one of the bottleneck problems for sustainably developing aquaculture and its effective preventing or curing methods are still rare. Our previous studies revealed that conjugated linoleic acid (CLA) could effectively decrease liver lipid accumulation in grass carp and miR-27 in liver is up regulated. Target gene prediction showed that Peroxisome proliferator-activated receptor gamma (PPARγ) ，one of key transcription factors on lipid metabolism, is an important target of miR-27. Thus, we propose the hypothesis that miR-27 regulates PPARγ maybe contribute to CLA decrease liver lipid accumulation in grass carp. To validate that, this project would study the targeting relationship between miR-27 and PPARγ, using some experimental techniques such as dual-luciferase reporter system, over/suppress expression of miRNA and Western blot, to explore the effect of miR-27 targeting regulate PPARγ on liver lipid accumulation in vivo and vitro. The results will be of great significance for us to clear the role and mechanism of miR-27 in CLA decreasing liver lipid accumulation in grass carp, and provide theoretical basis for using CLA to prevent the NALD of grass carp.

营养性脂肪肝是制约水产养殖业可持续发展的瓶颈问题，目前其有效的防治措施尚不多见，申请人所在课题组前期研究发现，共轭亚油酸（CLA）可显著降低草鱼肝脂积累。同时，miR-27在CLA组草鱼肝脏中显著上调，靶标基因预测得知，脂肪代谢的关键转录因子PPARγ为miR-27的重要靶标。据此假设：miR-27靶向调控PPARγ可能在CLA降低草鱼肝脂积累过程中发挥作用。因此，本项目拟以草鱼肝细胞系和活体为研究对象，从miR-27调控PPARγ表达水平为切入点，采用双荧光素酶报告系统、miRNA过表达/抑制表达、q-PCR、Western blot等技术探究miR-27对PPARγ的调控以及对细胞脂肪积累表型的影响，最终阐明miR-27在CLA降低草鱼肝脂积累中的作用及其分子机制，为草鱼营养性脂肪肝的防治提供理论依据。

营养性脂肪肝是制约水产养殖业可持续发展的瓶颈问题，目前其有效的防治措施尚不多见。我们前期研究共轭亚油酸（CLA）可显著降低草鱼肝脂积累，同时，miR-27在CLA组草鱼肝脏中显著上调，靶标基因预测得知，脂肪代谢的关键转录因子PPARγ为miR-27的重要靶标，提示miR-27可能参与了CLA降低草鱼肝脂积累的过程。当前研究以miR-27b（miR-27中的一个亚型）为研究对象，鉴定了miR-27b与PPARγ靶标关系，研究了高脂水平下草鱼肝脏脂质代谢、CLA对脂质代谢的调控及miR-27b在其中发挥的作用，为草鱼脂肪代谢的调控提供了新的理论指导。1）生物信息学预测靶点，构建双荧光素酶报告基因载体，发现miR-27b可以靶向PPARγ。2）通过腹腔注射miR-27b的激活剂（agormir）和拮抗剂（antagormirs），在活体水平研究miR-27b靶向调控PPARγ在CLA降低草鱼肝脂积累，研究发现CLA组miR-27b表达显著增强，miR-27b的激活剂（agormir）可显著抑制PPARγ表达，而miR-27b拮抗剂（antagormirs）组PPARγ表达显著增强。3）采用双荧光素酶报告系统验证过的miR-27b mimic和inhibitor，分别将miR-27b mimic或miR-27b inhibitor 转染草鱼肝细胞，模拟miR-27b过表达或抑制表达，在细胞水平研究miR-27b靶向调控PPARγ在CLA降低草鱼肝脂积累过程中的作用，研究发现CLA或miR-27b过表达均可显著抑制PPARγ表达，miR-27b inhibitor组PPARγ表达显著增强。本项目揭示miR-27在CLA发挥降低肝脂积累中的作用机制，有望为改善鱼类脂肪过度沉积现象提供新的作用靶点。