干扰Smad3促进山羊脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2020.03.008

Abstract

Abstract: The aim of this study was to clone goat Smad3, to clarify its tissue and cell expression profiles, and finally to elucidate the effect of interfering Smad3 gene on the differentiation of goat intramuscular and subcutaneous adipocytes. In this study, 5 one-week-old Jianzhou Big-ear goats in good physical condition were selected, slaughtered after fasted for 24 h, and the corresponding tissues and cells were sampled for testing. The cDNA sequence of goat Smad3 gene was cloned by RT-PCR and analyzed by bioinformatics. Real-time quantitative PCR (qPCR) was used to detect the tissue and cell temporal expression of Smad3 gene. siRNA targeting to Smad3 was designed and synthesized. Oil red O staining was used to detect the effect of interfering Smad3 on adipogenic differentiation of goat preadipocytes, and qPCR was used to detect the effect of interfering Smad3 on the expression levels of adipocyte differentiation marker genes (C/EBPα, C/EBPβ, LPL, SREBP1, AP2, PPARγ, Pref-1, KLF3, KLF4, KLF6, KLF7, KLF8, KLF9, KLF10 and KLF15), Smads-related genes (Smad2, Smad4, Smad7) and TGF-β1 mRNA, with exploring the possible mechanism. The results showed that the goat Smad3 gene was 1 449 bp in length, the CDS region sequence was 1 278 bp, encoding 425 amino acids. Smad3 was widely expressed in goat tissues with the highest expression level in the kidney (P<0.01); Smad3 expression was lowest in goat intramuscular and subcutaneous adipocytes at 36 h of induced differentiation, which was significantly lower than that in undifferentiated precursor adipocytes (P<0.01). The interference of Smad3 significantly promoted the accumulation of lipid droplets in intramuscular and subcutaneous adipocytes of goats, the expression level of adipocyte differentiation marker genes, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 were significantly increased (P<0.05), and the relative expression level of Pref-1 was extremely significantly decreased (P<0.01). At the same time, the interference of Smad3 gene down-regulated the relative expression levels of Smad2, Smad4 and Smad7 genes (P<0.05). The results indicate that interference of Smad3 promotes the differentiation of goat adipocytes by regulating the expression of adipocyte differentiation marker genes LPL, SREBP1, AP2, C/EBPα, C/EBPβ, Pref-1, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 cooperating with Smad2, Smad4 and Smad7.

Key words: goat, Smad3, gene cloning, RNA interference, intramuscular adipocytes, subcutaneous adipocytes

CLC Number:

CUI Sheng, LIN Yaqiu, XU Qing, ZHU Jiangjiang, WANG Yong. Interfering Smad3 Promotes Goat Adipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 475-489.

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Interfering Smad3 Promotes Goat Adipocyte Differentiation

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