MiR-449a has tumor-regulatory properties in different cancers, but its role in osteosarcoma had not been clearly understood. This research aimed to study the underlying mechanism of how miR-449a regulates osteosarcoma cells. The expression of miR-449a, cyclin D1, and pRb in osteosarcoma tissues and cultivated cells was assessed by qRT-PCR, western blot, and immunofluorescent assay. Dual-luciferase reporter assay was conducted to verify the target of miR-449a. Western blot, CCK-8, colony formation, and flow cytometry assays were applied to examine the regulatory effects of miR-449a on osteosarcoma cells and the molecular mechanism. MiR-449a and pRb expression was impeded whereas cyclin D1 expression was enhanced in osteosarcoma tissues and cells. Cyclin D1 was confirmed as a target of miR-449a. MiR-449a impeded the viability and proliferation of osteosarcoma cells and controlled the cell cycle through targeting cyclin D1 to reduce pRB phosphorylation. These findings provided evidence that miR-449a played an anticancer role in osteosarcoma cells by directly targeting cyclin D1 to prevent pRb from phosphorylation. Our study suggested that miR-449a might have the potential to become a new therapeutic biomarker involved in the diagnosis, prevention, and treatment of osteosarcoma.

中文翻译：

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MiR-449a 海绵细胞周期蛋白 D1 表达并抑制视网膜母细胞瘤蛋白对骨肉瘤的磷酸化。

MiR-449a 在不同的癌症中具有肿瘤调节特性，但其在骨肉瘤中的作用尚不清楚。本研究旨在研究miR-449a如何调节骨肉瘤细胞的潜在机制。通过 qRT-PCR、蛋白质印迹和免疫荧光测定评估 miR-449a、cyclin D1 和 pRb 在骨肉瘤组织和培养细胞中的表达。进行双荧光素酶报告基因测定以验证 miR-449a 的靶标。应用Western blot、CCK-8、集落形成和流式细胞仪检测miR-449a对骨肉瘤细胞的调节作用及其分子机制。MiR-449a 和 pRb 表达受到阻碍，而 cyclin D1 表达在骨肉瘤组织和细胞中增强。Cyclin D1 被确认为 miR-449a 的靶标。MiR-449a 阻碍骨肉瘤细胞的活力和增殖，并通过靶向 cyclin D1 降低 pRB 磷酸化来控制细胞周期。这些发现提供了证据，表明 miR-449a 通过直接靶向 cyclin D1 以防止 pRb 磷酸化，从而在骨肉瘤细胞中发挥抗癌作用。我们的研究表明，miR-449a 可能有可能成为参与骨肉瘤诊断、预防和治疗的新治疗性生物标志物。