miRNA-133a-5p Inhibits the Expression of Osteoblast Differentiation-Associated Markers by Targeting the 3′ UTR ofRUNX2

Abstract

Recent studies have recognized the involvement of microRNAs (miRNAs) in the development of osteoporosis, which regulate the balance between osteogenesis and osteoclasis. In this study, we investigated the regulation by miRNA-133a-5p on the osteoblast differentiation-associated markers in the mouse osteoblast-like MC3T3-E1 cells by RUNX2. First, we manipulated the miRNA-133a level in the MC3T3-E1 cells with 20 or 40 nM miR-133a-5p mimics, miR-133a-5p inhibitor, or scramble miRNA. Then, we quantified with real-time polymerase chain reaction (qRT-PCR) the expression of Collagen I, osteocalcin (OCN), and osteopontin (OPN) in the miR-133a-5p-manipulated MC3T3-E1 cells. And the confocal microscopy was also utilized to confirm the regulation by miR-133a-5p on the expression of the three molecules. We also investigated the extracellular matrix (ECM) mineralization and the alkaline phosphatase (ALP) activity in the miR-133a-5p-manipulated MC3T3-E1 cells. In addition, we explored the possible targeting by miR-133a-5p on RUNX2, which was a well-recognized promoter to osteoblast differentiation, with luciferase reporter, qRT-PCR, and Western blotting assay. Results demonstrated that the miRNA-133a-5p mimics markedly reduced, whereas the miRNA-133a-5p inhibitor significantly promoted the expression of Collagen I, OCN, and OPN, the ECM mineralization, and the ALP activity in MC3T3-E1 cells. The alignment analysis demonstrated a high homology between miRNA-133a-5p and the 3' UTR of RUNX2. Moreover, the luciferase reporter assay demonstrated that miRNA-133a-5p targeted the 3' UTR of RUNX2, and inhibited the expression of RUNX2 in both mRNA and protein levels. In conclusion, we identified the inhibition by miRNA-133a-5p to the expression of osteoblast differentiation markers, to the ECM mineralization, and to the ALP activity in MC3T3-E1 cells, by targeting the 3' UTR of RUNX2. Our study suggests that miRNA-133a-5p might be an important target to inhibit osteoblast differentiation in osteoporosis.

MicroRNA and Human Bone Health

“…As shown in other human studies, miR‐133a could promote osteoclast differentiation, and its level was correlated with low BMD in postmenopausal women . It was also shown that miR‐133a could hamper bone formation by targeting RUNX2 . MiR‐33, on the other hand, was involved in mechano‐induced osteoblast differentiation, but its association with BMD has yet to be reported.…”

The Role of MicroRNAs in Bone Metabolism and Disease

“…Thus, miR-140-3p regulated osteoblast differentiation by acting as a regulatory factor between Wnt3a and TGFβ3 signaling pathways [92]. According to the study, miR-133a-5p overexpression was shown to significantly lower the expression of osteoblast differentiation markers as well as ECM mineralization in MC3T3-E1 cells by targeting 3 UTR of Runx2 and reduce Runx2 expression at both mRNA and protein levels [93]. The expression of miR-92a-1-5p was decreased during MC3T3-E1 osteogenic differentiation, and overexpression could target β-catenin, a positive regulator of osteogenic differentiation, thereby inhibiting osteogenesis [94].…”

The roles of miRNA, lncRNA and circRNA in the development of osteoporosis

“…However, miR-375 suppresses osteogenic differentiation by directly targeting Runx2 and, in this process, it reduces the activity levels of key osteoblast markers, including osteocalcin, ALP, and collagen type I α1 (COL1A1) [32]. miRNA-133a-5p inhibits the expression of Runx2, at both mRNA and protein levels by targeting the 3′-UTR of Runx2 [33]. As an endogenous attenuator of Smad1, miR-100 was shown to inhibit BMP-induced osteoblast differentiation [34].…”