Department of Osteoarthritis, Jining No. 2 People's Hospital, Jining, Shandong 272049, P.R. China.
Department of Bone Oncology, Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou, Gansu 730050, P.R. China.
Mol Med Rep. 2019 Dec;20(6):4843-4854. doi: 10.3892/mmr.2019.10775. Epub 2019 Oct 29.
MicroRNAs serve an important role in the development of several diseases. Numerous genes regulate the skeletal muscle differentiation of C2C12 myoblasts. The role of miR‑760 in rheumatoid arthritis (RA) has not been reported, to the best of our knowledge. Therefore, the aim of the present study was to examine the role of miR‑760 in regulating skeletal muscle proliferation in RA. Potential genes functionally involved in the tarsal joint of a collagen‑induced RA model were identified using Gene Expression Omnibus. Reverse transcription‑quantitative PCR and western blot analyses were performed to determine the mRNA and protein expression levels. The proliferation, cell cycle progression and migration of C2C12 myoblasts were detected using Cell Counting Kit‑8, flow cytometry and wound‑healing assays, respectively. TargetScan was used to predict the potential target genes of miR‑760, and this was verified using a dual‑luciferase reporter assay. In the present study, myosin‑18b (Myo18b) expression was determined to be downregulated in the RA model. Silencing Myo18b decreased the proliferation, abrogated the cell cycle progression, and reduced the migration and differentiation of C2C12 myoblasts. Expression levels of cyclin‑dependent kinase 2, cyclin D1, matrix metalloproteinase (MMP)‑2, MMP‑9, myogenin and myosin heavy chain 6 were all decreased when Myo18b was silenced. Furthermore, overexpression of Myo18b induced opposing effects on C2C12 myoblasts. It was shown that Myo18b was a target gene of miRNA‑760. Overexpression of miR‑760 decreased proliferation, cell cycle progression, migration and differentiation in C2C12 myoblasts, and decreased the expression of Myo18b. The opposite results were observed when miR‑760 was downregulated. In conclusion, miR‑760 inhibited proliferation and differentiation by targeting Myo18b in C2C12 myoblasts. The results of the present study may contribute to understanding the mechanisms underlying RA skeletal muscle proliferation, and miR‑760/Myo18b may serve as potential targets for treating patients with RA.
微小 RNA 在多种疾病的发展中发挥着重要作用。许多基因调节 C2C12 成肌细胞的骨骼肌肉分化。据我们所知，miR-760 在类风湿关节炎（RA）中的作用尚未报道。因此，本研究旨在探讨 miR-760 在调节 RA 骨骼肌肉增殖中的作用。使用基因表达综合数据库鉴定与胶原诱导的 RA 模型跗关节相关的潜在功能基因。采用逆转录-定量 PCR 和 Western blot 分析检测 mRNA 和蛋白表达水平。使用细胞计数试剂盒-8、流式细胞术和划痕愈合试验分别检测 C2C12 成肌细胞的增殖、细胞周期进程和迁移。使用靶标扫描预测 miR-760 的潜在靶基因，并通过双荧光素酶报告基因试验进行验证。在本研究中，肌球蛋白 18b（Myo18b）在 RA 模型中的表达被确定下调。沉默 Myo18b 降低了 C2C12 成肌细胞的增殖，阻断了细胞周期进程，并减少了迁移和分化。沉默 Myo18b 后，细胞周期蛋白依赖性激酶 2、细胞周期蛋白 D1、基质金属蛋白酶（MMP）-2、MMP-9、肌生成蛋白和肌球蛋白重链 6 的表达水平均降低。此外，Myo18b 的过表达对 C2C12 成肌细胞产生了相反的影响。结果表明，Myo18b 是 miRNA-760 的靶基因。miR-760 的过表达降低了 C2C12 成肌细胞的增殖、细胞周期进程、迁移和分化，并降低了 Myo18b 的表达。下调 miR-760 时观察到相反的结果。总之，miR-760 通过靶向 C2C12 成肌细胞中的 Myo18b 抑制增殖和分化。本研究的结果可能有助于理解 RA 骨骼肌肉增殖的机制，miR-760/Myo18b 可能成为治疗 RA 患者的潜在靶点。
