Snail1对牛脂肪细胞增殖分化影响及作用机制的研究

doi:10.11843/j.issn.0366-6964.2023.12.012

Abstract

Abstract: This study aimed to explore the effect of Snail1 gene on the proliferation and differentia-tion of bovine adipocytes and its potential molecular mechanism. In this study, an adenovirus was used to overexpress the Snail1 gene in Qinchuan cattle adipocytes. Experimental and control groups were established, each with 3 biological replicates. The effect of Snail1 on the proliferation and differentiation of bovine adipocytes was investigated by flow cytometry, CCK-8, EdU staining, Oil Red O staining, triglyceride determination, qRT-PCR and Western blot. The signaling pathways and target genes were further screened by RNA-Seq and dual-luciferase reporter experiments. The results showed that overexpression of Snail1 inhibited cell proliferation (CCK-8) and reduced the proportion of positive cells at the S-phase (EdU, P<0.05). Combined with flow cytometry tests, the results indicated that the up-regulation of Snail1 led to G1/S phase arrest of the cell cycle. Furthermore, qRT-PCR and Western blot results showed that Snail1 inhibited the expression of CCNB1 and CCND2 (P<0.05). The results of Oil Red O staining and triglyceride detection on bovine adipocytes on day 6 of induced differentiation showed that Snail1 inhibited the adipogenesis and triglyceride formation of bovine adipocytes. qRT-PCR analysis revealed that the overexpression of Snail1 inhibited the gene expression of PPARγ (P=0.06), FABP4 (P<0.05), and LPL(P<0.05), while the expression level of PIK3R3 was significantly upregulated (P<0.01);The Western blot results domonstrated that expression of Snail1 significantly inhibited FABP4 protein expression. Further analysis through RNA-Seq revealed that the differentially expressed genes induced by Snail1 overexpression were predominantly enriched in signaling pathway such as the PPAR, ECM-receptor interaction, PI3K-AKT, MAPK, nicotine addiction, and insulin secretion. Based on FIMO target gene screening, combined RNA-Seq data analysis, and luciferase assay, it was confirmed that the transcription factor Snail1 potentially affects the differentiation process of bovine adipocytes by targeting the antagonist SFRP2 of the Wnt signaling pathway. The results suggested that Snail1 gene inhibited the proliferation and differentiation process of bovine adipocytes, and may be involved in the regulation of bovine adipocyte differentiation through the positive feedback loop of "Snail1/SFRP2-Wnt/β-catenin-GSK3 β".

Key words: cattle, Snail1 gene, adipocyte, proliferation and differentiation, RNA-Seq, target gene

CLC Number:

S823.8+1

ZHANG Wentao, LIU Chenyang, ZHU Binglin, LIU Li, TIAN Yuan, YAO Yuhang, CHENG Gong. Study on the Effects and Mechanisms of Snail1 on Proliferation and Differentiation of Bovine Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5008-5019.

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Study on the Effects and Mechanisms of Snail1 on Proliferation and Differentiation of Bovine Adipocytes

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