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MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1.

ABSTRACT: 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 ?-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/?-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/?-catenin signaling pathways in myogenesis.

SUBMITTER: Cai R

PROVIDER: S-EPMC6302170 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and <i>Wnt1</i>.

Cai Rui R Qimuge Naren N Ma Meilin M Wang Yingqian Y Tang Guorong G Zhang Que Q Sun Yunmei Y Chen Xiaochang X Yu Taiyong T Dong Wuzi W Yang Gongshe G Pang Weijun W

The Journal of biological chemistry 20181015 50

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 ...[more]

PMID: 30323063

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Project description:Background and purposeTrauma-induced osteonecrosis of the femoral head (TIONFH) is a major complication of femoral neck fractures. Degeneration and necrosis of subchondral bone can cause collapse, which results in hip joint dysfunction in patients. The destruction of bone metabolism homeostasis is an important factor for osteonecrosis. MicroRNAs (miRNAs) have an important role in regulating osteogenic differentiation, but the mechanisms underlying abnormal bone metabolism of TIONFH are poorly understood. In this study, we screened specific miRNAs in TIONFH by microarray and further explored the mechanism of osteogenic differentiation.DesignBlood samples from patients with TIONFH and patients without necrosis after trauma were compared by microarray, and bone collapse of necrotic bone tissue was evaluated by micro-CT and immunohistochemistry. To confirm the relationship between miRNA and osteogenic differentiation, we conducted cell culture experiments. We found that many miRNAs were significantly different, including miR-93-5p; the increase in this miRNA was verified by Q-PCR. Comparison of the tissue samples showed that miR-93-5p expression increased, and alkaline phosphatase (ALP) and osteopontin (OPN) levels decreased, suggesting miR-93-5p may be involved in osteogenic differentiation. Further bioinformatics analysis indicated that miR-93-5p can target bone morphogenetic protein 2 (BMP-2). A luciferase gene reporter assay was performed to confirm these findings. By simulating and/or inhibiting miR-93-5p expression in human bone marrow mesenchymal stem cells, we confirmed that osteogenic differentiation-related indictors, including BMP-2, Osterix, Runt-related transcription factor, ALP and OPN, were decreased by miR-93-5p.ConclusionOur study showed that increased miR-93-5p in TIONFH patients inhibited osteogenic differentiation, which may be associated with BMP-2 reduction. Therefore, miR-93-5p may be a potential target for prevention of TIONFH.

Dataset Information

MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1.

Publications

MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and <i>Wnt1</i>.

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