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

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 3864-3875.doi: 10.11843/j.issn.0366-6964.2024.09.012

miR-127调控绵羊骨骼肌细胞增殖分化及其转录因子PAX3筛选

贾宇航¹^,²(), 郭良富³, 张茹楠¹, 赵阿勇², 刘玉芳¹^,*(), 储明星¹^,*()

1. 中国农业科学院北京畜牧兽医研究所，畜禽生物育种全国重点实验室，北京 100193
2. 浙江农林大学动物科技学院·动物医学院，杭州 311300
3. 郓城县畜牧服务中心，郓城 274700

收稿日期:2024-03-04 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 刘玉芳,储明星 E-mail:15645551808@163.com;aigaiy@126.com;mxchu@263.net
作者简介:贾宇航(1999-)，男，黑龙江绥化人，硕士生，主要从事动物遗传育种研究，E-mail：15645551808@163.com
基金资助:
国家资助博士后研究人员计划(GZC20233053);财政部和农业农村部国家现代农业产业技术体系(CARS-38);中国农业科学院科技创新工程(CAAS-ZDRW202106);中国农业科学院科技创新工程(ASTIP-IAS13)

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

Yuhang JIA¹^,²(), Liangfu GUO³, Runan ZHANG¹, Ayong ZHAO², Yufang LIU¹^,*(), Mingxing CHU¹^,*()

1. State Key Laboratory of Animal Biotech Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2. College of Animal Science and Technology· College of Veterinary Medicine, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
3. Yuncheng County Animal Husbandry Service Center, Yuncheng 274700, China

Received:2024-03-04 Online:2024-09-23 Published:2024-09-27
Contact: Yufang LIU, Mingxing CHU E-mail:15645551808@163.com;aigaiy@126.com;mxchu@263.net

摘要/Abstract

摘要：

旨在探究miR-127对绵羊骨骼肌成肌细胞增殖与分化的影响，并通过鉴定miR-127上游核心启动子区域筛选调控其表达的转录因子。本研究以体外分离培养的小尾寒羊胎羊骨骼肌原代成肌细胞为试验材料，过表达或干扰miR-127后，采用RT-qPCR、EdU染色、流式细胞仪分析、免疫荧光染色等方法探究miR-127对绵羊成肌细胞增殖、凋亡和分化的影响。基于生物信息学分析和双荧光素酶报告试验预测并鉴定绵羊miR-127核心启动子区域及其转录因子。结果表明，在绵羊成肌细胞中过表达miR-127后，促进了细胞增殖相关基因PCNA、CDK4的表达(P < 0.01)；抑制了细胞凋亡相关基因Caspase3、BAX的表达(P < 0.05)；EdU结果显示，绵羊成肌细胞中过表达miR-127后，细胞阳性率显著提升(P < 0.05)；流式细胞仪分析显示，miR-127过表达可显著增加成肌细胞S期和G2期细胞比例并减少成肌细胞的凋亡。在细胞分化试验中，过表达miR-127显著增加了成肌细胞分化相关基因MYOD1和MYHC mRNA的表达水平(P < 0.05)，显著增加了MYHC的阳性肌管面积(P < 0.05)。抑制miR-127表达则出现与上述相反的结果。为进一步揭示调控miR-127表达的调控因子，双荧光素酶活性检测结果显示，miR-127上游1 500~1 800 bp(miR-127-P6)区段活性最高，推断其为miR-127的核心启动子区。生物信息学预测表明，在miR-127核心启动子区存在一个与转录因子PAX3的结合位点。在绵羊成肌细胞中过表达转录因子PAX3后，miR-127启动子活性和miR-127的表达均极显著增加(P < 0.01)。本研究表明，绵羊miR-127显著促进绵羊骨骼肌成肌细胞增殖分化，减少了成肌细胞凋亡，进而参与骨骼肌成肌细胞的发育过程；进一步研究发现，绵羊miR-127上游的1 500~1 800 bp是其核心启动子区，转录因子PAX3正向调节miR-127的转录。本研究为进一步探究绵羊肌肉生长发育性状的分子机制提供了理论参考。

关键词: 绵羊, 成肌细胞增殖分化, miR-127, 转录因子PAX3

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

中图分类号:

S826.2

贾宇航, 郭良富, 张茹楠, 赵阿勇, 刘玉芳, 储明星. miR-127调控绵羊骨骼肌细胞增殖分化及其转录因子PAX3筛选[J]. 畜牧兽医学报, 2024, 55(9): 3864-3875.

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.

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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调控绵羊骨骼肌细胞增殖分化及其转录因子PAX3筛选

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

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