miR-138靶向PGC-1α调控牦牛肌内前体脂肪细胞增殖及分化

doi:10.11843/j.issn.0366-6964.2022.10.016

Abstract

Abstract: The study aimed to analyze the regulatory action of miR-138 during the yak preadipocytes differentiation. In this study, miR-138 mimics and inhibitor were designed and synthesized to overexpress or suppress miR-138 in preadipocytes. The oil red O staining assay were performed to analyze the effects of miR-138 on lipid deposition, while CCK-8, scratch test assays and flow cytometry were performed to analyze the effects of miR-138 on proliferation. Furthermore, the potential target genes of bta-miR-138 in the process of lipid deposition were screened by bioinformatics analysis; And the mRNA expression levels of miR-138 target genes were detected by RT-qPCR technology, in addition, dual-luciferase report assay were used to verify the interaction between miR-138 and its target. The results showed that overexpression of miR-138 significantly reduced lipid deposition level compared with the control (NC) group (P<0.01), and significantly increased in lipid deposition when miR-138 suppression (P<0.01). RT-qPCR results showed that, overexpression of miR-138 also significantly inhibited the expression of adipose differentiation markers (PPARγ and c/EBPα, P<0.01), whereas the expression of PPARγ and c/EBPα were enhanced when miR-138 suppression (P<0.05). In addition, overexpression of miR-138 significantly down-regulated the mRNA expression of potential targets PTPN11, CREB1, ADCYAP1R1, PGC-1α and SNAP25 (P<0.05 or P<0.01), while the mRNA expression of MXLIP, SNAP25, PGC-1α and PTPN11 significantly in creased (P<0.05 or P<0.01) when miR-138 suppression. The results of CCK-8 and scratch test showed that the cell proliferation activity decreased significantly (P<0.05) when miR-138 overexpression 24 to 36 h, whereas the proliferation activity was increased after miR-138 inhibition. The flow cytometry analysis results indicated that cell cycle were arrested at G1 to S phase, and mRNA level of cell cycle-related genes CCND1, CCNB1 and the proliferation marker gene Ki67 were significantly decreased after miR-138 overexpression. A total of 263 common targets of miR-138 were predicted by bioinformatics analysis. GO annotation results showed that those target genes significantly enriched in "Positive regulation of transcription from RNA polymerase II promoter", "Nuclear chromatin", "Chromatin DNA binding", and "Type I pneumocyte differentiation", KEGG enrichment results showed that these target genes mainly participated in "Axon guidance", "Insulin resistance", "Insulin secretion" and "RNA degradation" pathways. The dual-luciferase report assay showed that co-transfection of miR-138 mimics and PGC-1α-Wt-PGL3-basic significantly reduced the cell fluorescence activity (P<0.01). The present results suggest that miR-138 can decrease the mRNA expression level of PGC-1α by binding its 3' UTR sequence, and inhibit the differentiation and lipid deposition of intramuscular preadipocyte in yak, thus decrease the proliferation of cell. The current study may help clarify the mechanism underlying yak meat characters formation at molecular level.

Key words: yak, miR-138, PGC-1α, preadipocyte, proliferation and differentiation

CLC Number:

S823.85

RAN Hongbiao, WANG Hui, CHAI Zhixin, WANG Jiabo, ZHANG Ming, CAI Xin, ZHONG Jincheng. miR-138 Regulates Proliferation and Differentiation of Intramuscular Preadipocyte by Targeting PGC-1α in Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3434-3447.

References 34

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miR-138 Regulates Proliferation and Differentiation of Intramuscular Preadipocyte by Targeting PGC-1α in Yak

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