Berberine regulates lipid metabolism via miR-192 in porcine oocytes matured in vitro

Abstract

Background: The berberine (Ber) is an isoquinoline alkaloid compound extracted from Rhizoma coptidis and has the effect that reduces adipose. MicroRNA-192 (miR-192) is related to fat metabolism. However, the relevant mechanism of berberine on lipid metabolism during in vitro maturation (IVM) of porcine oocytes remains unclear.

Objectives: In this study, we investigated the molecular mechanism by which berberine promotes the IVM and lipid metabolism of porcine oocytes via miR-192.

Methods: Ber was added to IVM medium of porcine oocytes. MiR-192 agomir, miR-192 antagomir and negative control fragment were microinjected into the cytoplasm of oocytes without Ber. Rates of oocyte IVM and embryonic development in each group were observed. The content of lipid droplets in IVM oocytes in each group was analyzed by Nile red staining. Expression levels of miR-192 and FABP3, SREBF1 and PPARG, were detected by qPCR and western blotting. The target genes of miR-192 were determined by luciferase reporter assays.

Results and conclusions: We found that Ber significantly increased the rate of oocytes IVM and blastocyst development, and decreased the area and numbers of lipid droplets in IVM oocytes. Ber significantly increased the expression of miR-192 in IVM oocytes, and significantly decreased the expression of SREBF1 and PPARG, which were target genes of miR-192. This study indicates that Ber promotes lipid metabolism in porcine oocytes by activating the expression of miR-192 and down-regulating SREBF1 and PPARG, thus, improving IVM of porcine oocytes.

Keywords: berberine; in vitro maturation; lipid metabolism; miR-192; porcine oocyte.

FIGURE 1

The results of each group by Nile Red dye. (a) The picture of each group oocyte by Nile Red dye. Scale bar indicates 75 μm. 1. Control; 2. negative control; 3. miR‐192 overexpression; 4. miR‐192 low expression and 5. Ber. (b) The effect of Ber and miR‐192 of lipid droplets area of porcine oocytes. The values denoted by different capital letters represent significant difference at p < 0.01 level, the same letters indicate no significant difference (p > 0.05)

FIGURE 2

The relative expression levels of miR‐192 in porcine IVM oocytes from different groups. The values denoted by different capital letters represent significant difference at p < 0.01 level, the same letters indicate no significant difference (p > 0.05)

FIGURE 3

Effect of Ber and miR‐192 on gene expression level related to lipid metabolism. (a) Effect of Ber and miR‐192 on the transcription level of genes related to lipid metabolism. (b) Effect of Ber and miR‐192 on the translation level of proteins related to lipid metabolism. The values denoted by different lowercase and capital letters represent significant difference at p < 0.05 level and p < 0.01 level respectively. And the same letters indicate no significant difference (p > 0.05)

FIGURE 4

The result of dual‐luciferase reporter gene assay of transfected vector plasmid. (a) The result of dual‐luciferase reporter gene assay of transfected FABP3 vector plasmid. (b) The result of dual‐luciferase reporter gene assay of transfected PPARG vector plasmid. (c) The result of dual‐luciferase reporter gene assay of transfected SREBF1 vector plasmid. PC, positive control; WT, wild‐type; MUT, mutant type; NC, negative control. **Indicates significant difference (p < 0.01), *Indicates significant difference (p < 0.05), no sign indicates no significant difference