miR-186-5p调控猪原代前体脂肪细胞增殖和分化的研究

doi:10.11843/j.issn.0366-6964.2021.05.011

Abstract

Abstract: The study aimed to explore the regulation and mechanism of miR-186-5p on the proliferation and adipogenic differentiation of porcine primary preadipocytes. The neck subcutaneous fat of 7-day-old healthy Mashen boar was collected and the primary preadipocytes were isolated and cultured. The primary preadipocytes were divided into 4 groups, which were transfected with miR-186-5p mimics (mimics) and its control group (mimics NC), miR-186-5p inhibitor (inhibitor) and its control group (inhibitor NC). CCK8 and scratch test were used to analyze the proliferation effect of porcine primary preadipocytes. Oil red O staining was used to detect the adipogenic differentiation ability. qPCR was used to detect the expression of proliferation and adipogenic differentiation related genes. The target genes of miR-186-5p were predicted, and the interaction between miR-186-5p and target genes was detected by dual luciferase reporter assay. When mimics were transfected into porcine primary preadipocytes, the expression of miR-186-5p significantly increased(P<0.01); meanwhile the cell proliferation ability and expression of proliferation related genes, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinases 4 (CDK4), and the target gene sirtuins 2 (SIRT2) significantly decreased(P<0.01); however, the adipogenic differentiation ability and expression of adipogenic differentiation related genes, peroxisome proliferators-activated receptor γ (PPARγ), sterol regulatory element binding transcription factor 1 (SREBF1), CCAAT enhancer binding protein β (C/EBPβ), fatty acid-binding protein 4 (FABP4) and lipoprotein lipase (LPL) significantly increased(P<0.01). When the primary preadipocytes were transfected with inhibitor, the expression of miR-186-5p significantly decreased(P<0.01); meanwhile the cell proliferation ability and expression of PCNA, CDK4 and SIRT2 significantly increased(P<0.05 or P<0.01); while the adipogenic differentiation ability and expression of PPARγ, SREBF1, C/EBPβ, FABP4 and LPL significantly decreased(P<0.01). Transfection of miR-186-5p mimics significantly inhibited the activity of luciferase in SIRT2-Wt-psiCHECK-2, but did not affect the activity of luciferase in SIRT2-Mut-psiCHECK-2. The results show that miR-186-5p can target the 3'UTR of SIRT2 and decrease the expression of SIRT2, which can inhibit the proliferation of Mashen pig primary preadipocytes and promote their adipogenic differentiation.

Key words: miR-186-5p, Mashen pigs, primary preadipocytes, proliferation, adipogenic differentiation, target gene

CLC Number:

S828.2

CAI Chunbo, LIU Min, YANG Yang, ZHANG Wanfeng, GAO Pengfei, CAO Guoqing, LI Bugao, GUO Xiaohong. Regulation of miR-186-5p on Proliferation and Differentiation of Porcine Primary Preadipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1247-1257.

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Regulation of miR-186-5p on Proliferation and Differentiation of Porcine Primary Preadipocytes

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