RESUMO
OBJECTIVE: To investigate whether Teriparatide (TP) contributed to the osteogenic differentiation of human marrow mesenchymal cells (hMSCs) through the regulation of miR-375, thereby alleviating osteoporosis (OP). PATIENTS AND METHODS: The expression levels of miR-375 in the serum and hMSCs of patients with OP were determined by quantitative real time-polymerase chain reaction (qRT-PCR). hMSCs were extracted from bone marrows of OP patients and underwent osteogenic differentiation for 1 day, 3 days, 7 days and 10 days, respectively. The mRNA levels of alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) in TP-treated hMSCs transfected with miR-375 mimics or negative control were detected by qRT-PCR. Western blot was conducted to determine the protein expression of RUNX2 in TP-treated hMSCs transfected with miR-375 mimics or negative control. Besides, the osteogenic capacity and mineralization capacity of hMSCs were evaluated by the detection of ALP activity, ALP staining and Alizarin red staining, respectively. Dual-Luciferase reporter gene assay was performed to verify the binding between RUNX2 and miR-375. Subsequently, RUNX2 expression was detected in hMSCs transfected with miR-375 mimics or inhibitor. Rescue experiments were finally performed to determine whether miR-375 was involved in TP-induced osteogenic differentiation by targeting RUNX2. RESULTS: MiR-375 remained at a high level in serum of OP patients, while gradually decreased with the prolongation of osteogenic differentiation in isolated hMSCs. TP induction increased the osteogenic and mineralization capacities of hMSCs, which were inhibited after miR-375 overexpression. Through Dual-Luciferase reporter gene assay, we confirmed the binding relationship between miR-375 and RUNX2. Besides, both mRNA and protein levels of RUNX2 were negatively regulated by miR-375. Finally, we verified that co-overexpression of miR-375 and RUNX2 in TP-induced hMSCs significantly enhanced the mineralization capacity compared to overexpression of miR-375 alone. CONCLUSIONS: Teriparatide promoted the osteogenic differentiation of hMSCs through miR-375/RUNX2 axis.
