The miR-6240 target gene Igf2bp3 promotes myoblast fusion by enhancing myomaker mRNA stability.

IF 9.2 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Yuxin Huang, Wei Wang, Xinhao Fan, Xiaoqin Liu, Weiwei Liu, Zishuai Wang, Yixing Li, Yalan Yang, Zhonglin Tang
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引用次数: 0

Abstract

Background: Myoblast fusion plays a crucial role in myogenesis. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) functions as an RNA N6-methyladenosine reader and exerts important roles in various biological processes. While our prior study suggested Igf2bp3 contributes to myogenesis, its molecular regulatory mechanism is largely unclear.

Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were used for gene expression analysis. siRNA and CRISPRi technologies were conducted to knockdown the expression of Igf2bp3. CRISPR/Cas9 technology was performed to knockout Igf2bp3. The Igf2bp3 overexpression vector was designed using the pcDNA3.1(+) vector. Immunofluorescence detection was employed for subcellular localization and cell differentiation analysis. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were conducted for cell proliferation and fusion detection. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized for regulatory mechanism analysis of Igf2bp3.

Results: The overexpression of Igf2bp3 enhances myoblast fusion while knockdown of Igf2bp3 blocks the formation of myotubes. miR-6240 promotes myoblast proliferation while preventing myoblast differentiation and fusion by targeting the 3' untranslated rgion (UTR) of Igf2bp3. Notably, the impacts of miR-6240 mimics on myoblast proliferation, differentiation, and fusion can be effectively counteracted by the overexpression of Igf2bp3. Moreover, our findings elucidate a direct interaction between Igf2bp3 and the myoblast fusion factor myomaker (Mymk). Igf2bp3 binds to Mymk to enhance its mRNA stability. This interaction results in increased expression of Mymk and heightened myoblast fusion.

Conclusions: Our study unveils Igf2bp3 as a novel post-transcriptional regulator of myoblast fusion through the miR-6240/Mymk axis, significantly contributing to our understanding of skeletal muscle development.

miR-6240靶基因Igf2bp3通过增强成肌细胞mRNA的稳定性来促进成肌细胞融合。
背景:成肌细胞融合在肌肉形成中起着至关重要的作用。胰岛素样生长因子2 mrna结合蛋白3 (Insulin-like growth factor 2 mRNA-binding protein 3, IGF2BP3)作为RNA n6 -甲基腺苷读取器,在多种生物过程中发挥重要作用。虽然我们之前的研究表明Igf2bp3参与肌肉发生,但其分子调控机制在很大程度上尚不清楚。方法:采用实时定量聚合酶链反应(RT-qPCR)和western blot技术进行基因表达分析。利用siRNA和CRISPRi技术敲除Igf2bp3的表达。采用CRISPR/Cas9技术敲除Igf2bp3。采用pcDNA3.1(+)载体设计Igf2bp3过表达载体。免疫荧光检测用于亚细胞定位和细胞分化分析。细胞计数试剂盒-8 (CCK-8)和5-乙基-2′-脱氧尿苷(EdU)检测细胞增殖和融合。采用双荧光素酶报告基因法和RNA免疫沉淀(RIP)法分析Igf2bp3的调控机制。结果:Igf2bp3过表达可促进成肌细胞融合,而Igf2bp3过表达可抑制成肌管的形成。miR-6240通过靶向Igf2bp3的3'未翻译区(UTR)促进成肌细胞增殖,同时阻止成肌细胞分化和融合。值得注意的是,miR-6240模拟物对成肌细胞增殖、分化和融合的影响可以通过Igf2bp3的过表达有效抵消。此外,我们的研究结果阐明了Igf2bp3与成肌细胞融合因子myomaker (Mymk)之间的直接相互作用。Igf2bp3结合Mymk增强其mRNA稳定性。这种相互作用导致Mymk表达增加和成肌细胞融合增强。结论:我们的研究揭示了Igf2bp3是一种通过miR-6240/Mymk轴调控成肌细胞融合的新型转录后调节因子,这对我们理解骨骼肌发育有重要意义。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Cellular & Molecular Biology Letters
Cellular & Molecular Biology Letters 生物-生化与分子生物学
CiteScore
11.60
自引率
13.30%
发文量
101
审稿时长
3 months
期刊介绍: Cellular & Molecular Biology Letters is an international journal dedicated to the dissemination of fundamental knowledge in all areas of cellular and molecular biology, cancer cell biology, and certain aspects of biochemistry, biophysics and biotechnology.
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