Canonical Wnt signaling activates miR-34 expression during osteoblastic differentiation

The canonical Wnt signaling pathway is crucial for the regulation of bone mass in humans and for the development of osteoblasts. MicroRNAs (miRs) represent a class of non‑coding RNAs, ~22 nucleotides in length, that regulate gene expression by targeting mRNAs for cleavage or translational repression. Several previous studies have demonstrated the involvement of miRNAs in modulating gene expression in osteoblasts and regulating osteoblast differentiation. In the present study, microRNA profiling was conducted using Wnt3a‑C2C12 cells; C2C12 cells were transfected with a Wnt3a expression plasmid to activate canonical Wnt signaling. miR‑34b‑5p and miR‑34c were identified to be upregulated by the activation of canonical Wnt signaling in C2C12 cells. Expression of mature miR‑34b/c increased from low levels at day 0 to maximum levels at day 28 of MC3T3‑E1 cell differentiation. To analyze the effects of these miRNAs on osteoblast differentiation, an antisense inhibitor was transfected into MC3T3‑E1 cells and osteoblast‑related gene expression was investigated. Knockdown of miR34b/c enhanced osteocalcin mRNA expression; however, alkaline phosphatase mRNA expression and activity were decreased by miR34b/c inhibition. These results indicated that miR‑34b/c regulates gene expression by targeting regulators of the osteogenic pathways and thereby contributes to osteoblast differentiation.