MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1
MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1
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MicroRNA-664-5p 通过靶向血清反应因子和 Wnt1 促进成肌细胞增殖并抑制成肌细胞分化
DOI：
10.1074/jbc.ra118.003198
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发表时间：
2018-12-14
影响因子：
4.8
通讯作者：
Pang, Weijun
中科院分区：
文献类型：
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作者：
Cai, Rui;Qimuge, Naren;Pang, Weijun
microRNA（miRNAs）是一类在转录后水平调控基因表达的非编码RNA，参与骨骼肌的形成、维持和功能的调控。通过miRNA测序和生物信息学分析，我们发现miR-664- 5 p在荣昌猪背最长肌中差异表达。然而，miR-664- 5 p调控肌生成的分子机制仍不清楚。本研究通过流式细胞术、5-乙炔基-2 ′-脱氧尿苷染色、细胞计数和免疫荧光检测发现，转染miR-664- 5 p mimics的C2 C12小鼠成肌细胞增殖能力明显增强，其机制可能是通过增加S期和G2期细胞比例以及上调细胞周期相关基因的表达。此外，miR-664- 5 p通过下调成肌基因表达来抑制成肌细胞分化。相反，miR-664- 5 p抑制剂抑制成肌细胞增殖并促进成肌细胞分化。从机制上讲，使用双荧光素酶报告基因实验，我们证明了miR-664- 5 p直接靶向血清反应因子（SRF）和Wnt 1 mRNA的3′-UTR。我们还观察到miR-664- 5 p分别在成肌细胞增殖和成肌分化过程中抑制SRF和Wnt 1的mRNA和蛋白水平。此外，miR-664- 5 p对成肌细胞增殖的激活作用通过SRF过表达而减弱，并且miR-664- 5 p通过减少核β-catenin的积累而抑制肌原性分化。值得注意的是，miR-664- 5 p对成肌分化的抑制作用通过Wnt 1蛋白（Wnt/β-连环蛋白信号通路的关键激活剂）处理而被消除。总的来说，我们的研究结果表明，miR-664- 5 p控制肌发生中的SRF和经典Wnt/β-catenin信号通路。
MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2′-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G2-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3′-UTR of serum response factor (SRF) and Wnt1 mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of SRF and Wnt1 during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by SRF overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear β-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/β-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/β-catenin signaling pathways in myogenesis.