MicroRNA-3064-3p regulates the differentiation of cementoblasts through targeting DKK1.

Abstract

MicroRNAs (miRNAs) are short, noncoding RNAs that interfere with translation of target mRNAs and thereby play a pivotal role in a variety of biological processes. Cementoblasts are the cells that build up cementum. They share a similar gene expression pattern with osteoblasts. Recent studies have suggested that miRNAs are able to control osteoblast-mediated bone formation. However, the effects of miRNA on cementoblast differentiation still remain unsolved. Herein, we wanted to elucidate the role of miR-3064-3p in cementoblast differentiation. A miRNA microarray was operated to explore the miRNA expression patterns during cementoblast differentiation. miR-3064-3p agomir/antagomir was used to promote or inhibit, respectively, the expression of miR-3064-3p. In order to measure the differentiation level of cementoblasts, quantitative RT-PCR (qRT-PCR), Alizarin red staining, and assessment of alkaline phosphatase activity were performed. Luciferase assays, qRT-PCR, and western blotting were used to identify the target gene of miR-3064-3p. miR-3064-3p showed persistently decreased expression during cementoblast differentiation. Overexpression of miR-3064-3p suppressed cementoblast differentiation, while inhibition of miR-3064-3p promoted cementoblast differentiation. Target prediction-analysis tools and dual-luciferase assay identified Dickkopf WNT signaling pathway inhibitor 1 (DKK1) as a direct target of miR-3064-3p. Results from qRT-PCR and western blotting showed that inhibition of miR-3064-3p led to a remarkable increase in DKK1/Dickkopf related protein 1 (Dkk-1) expression. In addition, pretreatment with recombinant Dickkopf related protein 1 (Dkk-1) rescued the miR-3064-3p-mediated suppression of cementoblast differentiation. This study demonstrates, for the first time, that miR-3064-3p suppresses cementoblast differentiation via the regulation of DKK1.