调控牛CEBPA基因表达的miRNA筛选及对肌内前体脂肪细胞增殖和分化的影响

doi:10.11843/j.issn.0366-6964.2021.006

Abstract

Abstract: To screen and verify the miRNA regulating the expression of cattle CEBPA (CCAAT/enhancer binding protein alpha, CEBPA) and their effects on the proliferation and differentiation of intramuscular precursor adipocytes. In this study, multiple online softwares were used to screen miRNA with potential regulation relationships with CEBPA genes. qRT-PCR technology was used to detect CEBPA gene expression levels in various tissues of Guanling cattles. By constructing CEBPA-3'UTR-WT and CEBPA-3'UTR-MUT dual luciferase reporter vectors, and then it was used to verify the regulatory relationship between candidate miRNAs and CEBPA genes. After Guanling cattle intramuscular preadipocytes were isolated and cultured, qRT-PCR, Western blot, Oil Red O staining and CCK-8 were used to explore the effect of miRNA regulating CEBPA on the differentiation and proliferation of bovine intramuscular preadipocytes. Bioinformatics analysis revealed that bta-miR-23a, bta-miR-23b-3p, bta-miR-181a, bta-miR-181b, bta-miR-181c, bta-miR-181d and CEBPA genes had potential regulating relationship. CEBPA gene was expressed in the visceral tissues, muscle tissues, fat, hypothalamus and part of the reproductive organs of Guanling cattles, the expression level in the longissimus dorsi of 2-year-old cattle was significantly higher than that in 1 day old calf (P<0.01). The results of the dual luciferase reporter carrier system indicated that bta-miR-23a, bta-miR-23b-3p, and bta-miR-181a could regu-lation the cattle CEBPA gene 3'UTR; Cell test results showed that the expression level of CEBPA gene in the differentiation of cattle intramuscular precursor adipocytes gradually increased with time, and bta-miR-23a, bta-miR-23b-3p and bta-miR-181a showed extremely significantly higher expression levels at 0 d differentiation than other periods (P<0.01), the expression trends of bta-miR-23b-3p and bta-miR-181a were opposite with CEBPA gene during the whole differentiation period; Overexpression of miR-23a, miR-23b-3p, miR-181a could extremely significantly reduce CEBPA gene mRNA and protein expression (P<0.01), and reduce the content of lipid droplets in cattles intramuscular precursor adipocytes. bta-miR-23a, bta-miR-23b-3p, bta-miR-181a could promote cell proliferation in stages. The results indicate that bta-miR-23a, bta-miR-23b-3p, bta-miR-181a can promote the proliferation of intramuscular precursor adipocytes of cattles in stages, and indirectly affect the differentiation of this cell by directly negatively regulating the CEBPA expression.

Key words: CEBPA, miRNA, intramuscular precursor adipocytes, cell differentiation

CLC Number:

S823.2

HUANG Mingjie, CHEN Xiang, ZHANG Yong, CHEN Wei, LI Jun, LU Jing, ZHANG Xiong, HE Qi, WU Yu. Screening of miRNA Regulating the Expression of Cattle CEBPA Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Precursor Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 52-65.

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Screening of miRNA Regulating the Expression of Cattle CEBPA Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Precursor Adipocytes

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