miR-145在黄斑蓝子鱼HUFA合成调控中的作用及机制研究

To clarify the regulatory mechanism of highly unsaturated fatty acids (HUFA) biosynthesis in fish may be helpful to resolve the problem that the formula feed for marine fish is overwhelmingly dependent on fish oil. In recent years, we have been engaged on the research of the metabolism of HUFA biosynthesis in Siganus canaliculatus, and firstly reported that miR-17 was involved in the regulation of HUFA biosynthesis via targeting a fatty acyl desaturase (delta-4 fad), while miR-33 was via cooperating with the transcription factor SREBP in Siganus canaliculatus. Recently, we have found that there was a binding site of miR-145 in the 3’-untranslated region (3’UTR) of HNF4α. And it was demonstrated that an interaction really existed between the 3’UTR of HNF4α and miR-145. We previously demonstrated that HNF4α was a transcription factor that plays an important role in the regulation of HUFA biosynthesis in Siganus canaliculatus. This suggested that the regulation pathway of miR-145 on the HUFA biosynthesis might be different from miR-17 and miR-33. Therefore, we aim to investigate the effects of miR-145 on the HUFA biosynthesis via targeting HNF4α gene as well as its downstream gene expression network. Our study may illuminate the regulatory mechanism of miR-145 on HUFA biosynthesis in Siganus canaliculatus. The results will be helpful to clarify the regulatory mechanism of HUFA biosynthesis in fish and provide new ideas to develop the techniques for fish to increase the capability to produce much more HUFA.

弄清鱼类高不饱和脂肪酸(HUFA)合成调控机制有助于解决海水鱼配合饲料对鱼油依赖性过强的问题。近年来本课题组在研究黄斑蓝子鱼HUFA合成代谢调控中，首次报道miR-17通过靶向作用∆4fad基因、miR-33通过与转录因子SREBP共表达参与HUFA的合成调控。最近，我们发现转录因子HNF4α基因的3’非编码区存在miR-145的结合位点，且实验证明它们之间存在互作关系。而前期我们已证实HNF4α参与HUFA的合成调控，这提示miR-145可能以不同于miR-17/33的作用方式调控HUFA的合成。本项目拟对miR-145参与HUFA合成调控的作用机制进行深入研究，包括miR-145靶向作用HNF4α参与HUFA合成代谢的功能确认，miR-145对HNF4α下游基因的调控作用及调控网络分析等。研究成果可为全面揭示鱼类HUFA合成调控机制提供新资料，为研发提高鱼体HUFA合成能力提供新思路。

本项目以黄斑蓝子鱼（Siganus canaliculatus）为研究对象，研究了miR-145通过靶向作用转录因子Hnf4α，参与蓝子鱼高不饱和脂肪酸（HUFA）合成代谢的调控作用及其分子机制，开展的具体研究内容及取得的主要结果有：1.在体外蓝子鱼肝细胞系SCHL上开展敲低与过表达miR-145实验，进一步验证了hnf4α是miR-145直接作用的靶基因；2. 通过在SCHL上敲低hnf4α，对其下游调控基因网络进行挖掘与鉴定，发现除了HUFA合成关键酶基因∆4fads2、∆6∆5 fads2和elovl5是Hnf4α转录调控的靶基因外，转录因子Srebp1也可能是其间接调控的靶基因之一；3. 通过在SCHL上敲低miR-145后进行脂肪酸底物亚麻酸的孵育，及在敲低miR-145的基础上进一步敲低hnf4α后，发现miR-145具有调控肝细胞HUFA合成的作用，且可能是通过靶向抑制hnf4α及其下游HUFA合成相关基因的表达，从而抑制蓝子鱼肝细胞HUFA的合成；4. 通过蓝子鱼腹腔注射miR-145的抑制剂antagomir21天后，检测各组织靶基因及下游相关基因的表达及脂肪酸的组成，结果进一步验证了miR-145是通过靶向抑制hnf4α及其下游HUFA合成相关基因的表达，进而抑制蓝子鱼体内HUFA的合成。此外，除了miR-145，我们同时还研究了miR-146a、miR-24、miR-33、miR-26a及miR-15/16在蓝子鱼HUFA合成调控中的功能及其作用机制，并发表了论文。通过本项目的研究，从分子水平上阐明了miRNA通过靶向HUFA合成相关基因的表达参与黄斑蓝子鱼HUFA合成代谢的调控作用及可能机制，为全面揭示鱼类HUFA合成代谢调控机制提供了新的资料，也可为研发提高鱼类HUFA内源性合成能力的方法提供新的思路。