Melatonin‑induced miR‑181c‑5p enhances osteogenic differentiation and mineralization of human jawbone‑derived osteoblastic cells

• Published in: Molecular medicine reports Vol. 22; no. 4; pp. 3549 - 3558
• Main Authors: Murodumi, Hiroshi, Shigeishi, Hideo, Kato, Hiroki, Yokoyama, Sho, Sakuma, Miyuki, Tada, Misato, Ono, Shigehiro, Rahman, Mohammad Zeshaan, Ohta, Kouji, Takechi, Masaaki
• Format: Journal Article
• Language: English
• Published: Greece Spandidos Publications 01-10-2020; Spandidos Publications UK Ltd
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - metabolism; Analysis; Antibodies; Bone morphogenetic proteins; Calcification; Cbfa-1 protein; Cell differentiation; Cell Differentiation - drug effects; Cell Line; Chromosome 5; Core Binding Factor Alpha 1 Subunit - genetics; Core Binding Factor Alpha 1 Subunit - metabolism; DNA microarrays; EDTA; Enzymatic activity; Enzymes; Experiments; Female; Fibroblast growth factor 2; Fibroblast growth factors; Gene expression; Gene Expression Profiling; Growth factors; Humans; Jaw - chemistry; Jaw - cytology; Jaw - drug effects; Kinases; Laboratories; Mandible; Melatonin; Melatonin - pharmacology; MicroRNA; MicroRNAs; MicroRNAs - genetics; Mineralization; miRNA; Notch2 protein; Oligonucleotide Array Sequence Analysis; Osteoblastogenesis; Osteoblasts; Osteoblasts - chemistry; Osteoblasts - cytology; Osteoblasts - drug effects; Osteogenesis; Pharmaceutical industry; Phosphatases; Proteins; Reagents; Scientific equipment industry; siRNA; Transfection; Young Adult; United States; Germany
• ISSN: 1791-2997; 1791-3004
• DOI: 10.3892/mmr.2020.11401

Our previous study revealed that treatment with a combination of fibroblast growth factor‑2 and melatonin (MEL) synergistically augmented osteogenic activity and mineralization of MC3T3‑E1 mouse preosteoblast cells. Thus, the objective of the present study was to assess the effect of MEL on osteogenetic characteristics in human osteoblastic cells. Human jawbone‑derived osteoblastic (hOB) cells were isolated from mandibular bone fragments. RUNX family transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) enzyme activity and the mineralization ability of hOB cells in the presence of MEL were evaluated. Microarray analysis was also performed to assess the expression of MEL‑induced microRNAs (miRNAs/miRs) in hOB cells. Treatment with MEL significantly enhanced Runx2 expression, ALP activity and mineralization staining. However, this effect was significantly reduced following transforming growth factor‑β1 treatment. In total, 124 miRNAs were differentially expressed in MEL‑treated hOB cells, compared with untreated cells. Of the upregulated miRNAs, miR‑181c‑5p exhibited the largest fold change. Runx2 mRNA expression and mineralization staining in the presence of MEL were significantly reduced following transfection with a miR‑181c‑5p inhibitor. In addition, transfection with miR-181c-5p mimics significantly increased Runx2 expression and mineralization staining. These results suggested that MEL‑induced miR‑181c‑5p was involved in osteogenic differentiation and mineralization of hOB cells. Using TargetScan, a putative miR‑181c‑5p binding site was identified in the Notch2 gene. Moreover, Notch2 mRNA and protein expression levels in hOB cells were significantly reduced following transfection with miR‑181c‑5p mimics, confirming Notch2 as a target gene for miR‑181c‑5p. Notch2 siRNA knockdown significantly increased Runx2 expression and mineralization staining, which suggested that Notch2 may negatively regulate osteogenic differentiation of hOB cells by downregulating Runx2. In conclusion, MEL‑induced expression of miR‑181c‑5p enhanced osteogenic differentiation and calcification of hOB cells.