MiR-449a Sponges Cyclin D1 Expression and Suppresses Phosphorylation of Retinoblastoma Protein against Osteosarcoma.

Abstract

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.