miR-127调控绵羊骨骼肌细胞增殖分化及其转录因子PAX3筛选

doi:10.11843/j.issn.0366-6964.2024.09.012

Abstract

Abstract:

The aim of this study was to investigate the effects of miR-127 on the skeletal myoblast proliferation and differentiation in sheep, and to screen for transcription factors that regulate the expression of miR-127 by identifying its upstream core promoter region. In this study, primary skeletal muscle myoblasts of small-tailed Han fetal sheep were used as experimental materials. After overexpression or inhibition of miR-127, the RT-qPCR, EdU staining, flow cytometry analysis, and immunofluorescence staining were used to detect the effects of miR-127 on the proliferation, apoptosis and differentiation of sheep myoblasts. The bioinformatics analysis and dual luciferase reporting assay were used to predict and identify the sheep miR-127 core promoter region and its transcription factors. The results showed that overexpression of miR-127 in sheep myoblasts promoted the expression of cell proliferation related genes PCNA and CDK4 (P < 0.01), and inhibited the expressions of cell apoptosis related genes Caspase3 and BAX (P < 0.05). EdU results showed that the positive rate of sheep myoblasts was significantly increased after miR-127 overexpression (P < 0.05). Flow cytometry analysis showed that overexpression of miR-127 significantly increased the proportion of S and G2 phase myoblasts and decreased the apoptosis of myoblasts. In the cell differentiation assay, overexpression of miR-127 significantly increased the mRNA expression levels of myoblast differentiation related genes MYOD1 and MYHC (P < 0.05), and significantly increased the MYHC positive myotube area (P < 0.05). The opposite result was found after miR-127 inhibition. In order to further reveal the regulatory factors regulating the expression of miR-127, the dual luciferase activity assay showed that the 1 500-1 800 bp (miR-127-P6) region upstream of miR-127 had the highest activity, which was inferred to be the core promoter region of miR-127. Bioinformatics predictions revealed a site in the core promoter region of miR-127 that bound to the transcription factor PAX3. Overexpression of the transcription factor PAX3 in sheep myoblasts significantly increased the promoter activity and the expression of miR-127 (P < 0.01). This study showed that sheep miR-127 significantly promoted the proliferation and differentiation of skeletal muscle myoblasts and reduced their apoptosis, which participated in their development process. Further studies showed that 1 500-1 800 bp upstream was the core promoter region of sheep miR-127, and the transcription factor PAX3 positively regulated the transcription of miR-127. This study provided a theoretical reference for further exploring the molecular mechanism of sheep muscle growth and development.

Key words: sheep, myoblast proliferation and differentiation, miR-127, transcription factor PAX3

CLC Number:

S826.2

Yuhang JIA, Liangfu GUO, Runan ZHANG, Ayong ZHAO, Yufang LIU, Mingxing CHU. miR-127 Regulated the Proliferation and Differentiation of Sheep Skeletal Myoblasts and Its Transcription Factor PAX3 Screening[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3864-3875.

Figures/Tables 9

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载体名称 Vector name	载体序列(5′→3′) Vector sequence
miR-127 mimics	F: AUCGGAUCCGUCUGAGCUUGGCU R: CCAAGCUCAGACGGAUCCGAUUU
miR-127 inhibitor	F: AGCCAAGCUCAGACGGAUCCGAU

基因名称 Gene name	基因ID GenBank ID	序列(5′→3′) Sequence	产物大小/bp Product size
PCNA	XM_004014340.5	F: TGCAGATGTACCCCTTGTGTGT R: CATCCTCGATCTTGGGAGCC	83
CDK4	XM_012158548.4	F: CAGTGTACAAGGCCCGTGAT R: GACGTCCATGAGCCTGACAA	176
BAX	XM_027978592.3	F: GCCCTTTTGCTTCAGGGTTTC R: TCGGAAAACATTTCAGCCGC	236
Caspase3	XM_060406953.1	F: CCAGGAGTGGAATCCAGCAG R: CGACAGGCCATGCCAGTATT	250
MYOD1	NM_001009390.1	F: TGCACGTCTAGCAACCCAAA R: GCTGTAGTCCATCATGCCGT	240
MYHC	XM_004010325.3	F: TCGTCAAGGCCACAATTTG R: CTGCTGCAACACCTGGTCCT	101
β-actin	XM_060405599.1	F: CTTCCAGCCTTCCTTCCTGG R: GGGGCGCGATGATCTTGA	215
miR-127	-	F: CCGATCGGATCGTCTGAGCTTGGCT R: 通用引物
U6	-	F: CAAGGATGACACGCAAATTCG R: 通用引物

片段 Fragment	序列(5′→3′)Sequence
miR-127-1	F: GTTGCTGGTTGATGAGTTGGTT R: CGAGGGCGATGATTGTGTC
miR-127-2	F: AAGACGACACAATCATCGCC R: GAAACCTCATGGAATTCGCC
miR-127-3	F: AGTCCGCTTGTCCATTTGG R: AGAGCCCTCTGAGCTTCAGC

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miR-127 Regulated the Proliferation and Differentiation of Sheep Skeletal Myoblasts and Its Transcription Factor PAX3 Screening

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