Effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells
Hao Sun1, Zhixu Liu1, Biao Li1
1Department of Oral and Maxillofacial Surgery, Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, P.R. China.
Abstract:
Distal-less genes (DLX) play important roles in regulating organism development. DLX2 is crucial for the differentiation and development of the primordium, which determines the subsequent development and phenotype of the maxillofacial skeletal patterns, and is the primary candidate gene that regulates the development of the first branchial arch. The aim of the present study was to investigate the effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells in vitro. A DLX2-expression retrovirus vector was constructed by subcloning with a murine stem cell virus (MSCV) and verified by sequencing. MC3T3-E1 cells were transfected with pMSCV-DLX2 and stable clones were selected with puromycin. The mRNA and protein expression levels of DLX2 were determined using quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. In addition, the expression levels of the osteogenic biomarkers, alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor (RUNX)2 and Msh homeobox (MSX)2, were assessed by quantitative PCR. ALP detection and Alizarin red staining were conducted to evaluate the effect of DLX2 overexpression on osteogenic differentiation. The data were analyzed by analysis of variance using the Student-Newman-Keuls method. Successful pMSCV-DLX2 construction, as verified by direct sequencing, enabled DLX2 overexpression in vitro. Enhanced ALP activity and Alizarin red staining were observed in the MC3T3-E1-DLX2 cells when compared with the control group. During osteogenic induction, DLX2 overexpression was demonstrated to upregulate ALP and MSX2 expression at the early stage and OCN expression at the late stage, while no statistically significant difference was observed in RUNX2 expression when compared with the control group. Therefore, DLX2 overexpression in vitro induced the osteogenic differentiation of MC3T3-E1 cells via upregulating bone formation-associated genes, such as ALP and MSX2.
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