Effects of lncRNA DANCR on proliferation and differentiation of osteoblasts by regulating the Wnt/β-catenin pathway.

Abstract

To measure the expression level of long non-coding ribonucleic acids (lncRNAs) differentiation antagonizing non-protein coding RNA (DANCR) in serum of patients with fracture and investigate its influences on the proliferation and differentiation of osteoblasts. Serum samples were collected from 44 fracture patients treated in our hospital and 24 healthy people receiving physical examination in our hospital. Then, reverse transcription-polymerase chain reaction (RT-PCR) technique was used to detect the expression of lncRNA DANCR in the serum of patients with fracture and healthy subjects. MC3T3-E1 mouse osteoblast cell line with stably-knocked out DANCR was further established using small interfering RNAs (siRNAs), and the effect of DANCR knockout on the proliferation of osteoblasts was determined using cell counting kit-8 (CCK-8). At the same time, 5-Ethynyl-2'-deoxyuridine (EdU) staining assay was performed to detect the percentage of EdU-positive cells in osteoblasts in control group and DANCR knockout group. In addition, the mRNA levels of differentiation-related genes including Runt-related transcription factor 2 (Runx2), Collagen1α1, osteocalcin (OC) and osterix (OSX) were detected via RT-PCR, and the protein level of Runx2 was measured through Western blotting. Moreover, osteoblasts were cultured with osteogenic medium for 14 d, and then alizarin red staining and alkaline phosphatase (ALP) staining assays were carried out to examine the differentiation of these osteoblasts. Lastly, Western blotting technique was employed to analyze the expression of the Wnt/β-catenin signaling pathway. The expression of lncRNA DANCR was significantly increased in the serum of fracture patients (p<0.05). The results of in-vitro cell experiments showed that the intervention of DANCR with siRNA was able to clearly promote the proliferation and differentiation of MC3T3-E1 osteoblast cell line. According to the results of Western blotting, DANCR promoted the apoptosis and proliferation, which was mediated by the activated Wnt/β-catenin signaling pathway in osteoblasts. LncRNA DANCR inhibition can facilitate the proliferation and differentiation of osteoblasts by activating the Wnt/β-catenin signaling pathway in osteoblasts. Therefore, DANCR is expected to be a new target promoting fracture healing.