Abstract:

The study aimed to explore the regulatory effect of miR-375-3p on porcine adipocyte generation and its action mechanism targeting Fam229a. In this study, 10 healthy and disease-free boars of 30 days old were selected to be reared under the same conditions. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression distribution of miR-375-3p in the heart, liver, spleen and other tissues and the expression levels at different stages of adipogenic differentiation of adipocytes. Using miRNA mimics and miRNA inhibitors to overexpress and interfere with miR-375-3p, qPCR was employed to detect the effect of miR-375-3p on the expression of key adipogenic genes CEBPα, PPARγ, and FABP4. Oil red O staining and statistical analysis were used to assess the adipogenic capacity. Bioinformatics analysis and dual luciferase reporter assays were used to predict and identify Fam229a as a downstream target of miR-375-3p, and the function of Fam229a was subsequently validated. Finally, the downstream functions of Fam229a were predicted using PSIPRED software, Phyre2.0 software, and the STRING database, and qPCR was employed to detect the expression of early adipogenic key genes CEBPα, CEBPβ, ZFP423, and ZFP521 regulated by Fam229a. All of the above experimental methods were performed in triplicate to ensure biological reproducibility. The results showed that miR-375-3p was widely expressed in various tissues of pigs, with relatively high expression levels in lung and perirenal lipids. The temporal expression profile showed that the expression of miR-375-3p decreased first and then increased during the differentiation of porcine primary precursor adipocytes. After overexpression of miR-375-3p, the lipid droplets of porcine primary precursor adipocytes increased significantly, and the mRNA levels of adipocyte differentiation marker genes CEBPα, PPARγ and FABP4 increased significantly (P < 0.01). However, the results were reversed after interfering with miR-375-3p. In order to explore the action mechanism of miR-375-3p, a binding effect between miR-375-3p and Fam229a was found through qRT-RCR, binding site prediction and dual luciferase assay. Overexpression of miR-375-3p could significantly inhibit the expression of Fam229a (P < 0.05). After interference, the opposite result is indicated. Tissue expression profile showed that Fam229a was relatively high in liver and perirenal lipids. The temporal expression profile showed that the expression of Fam229a increased first, then decreased and then increased during the differentiation of porcine primary precursor adipocytes. After overexpression of Fam229a, the lipid drops were significantly decreased, and the mRNA levels of adipocyte differentiation marker genes CEBPα and PPARγ were significantly decreased (P < 0.05), while the results were opposite after interfering with Fam229a. The downstream mechanism of Fam229a regulating adipogenic differentiation was predicted by qRT-RCR technique and bioinformatics. This study results show that miR-375-3p can promote the adipogenic differentiation of porcine primary precursor adipocytes, and its target gene Fam229a inhibits adipogenic differentiation, revealing the regulation mechanism of adipogenic differentiation by miR-375-3p targeting Fam229a, and enriching the molecular regulatory network of miRNA.

Key words: pigs, adipocyte differentiation, miR-375-3p, Fam229a