Depletion of PRDM9 inhibited the osteogenic differentiation potential of periodontal ligament stem cells
10.3760/cma.j.issn.1002-0098.2019.12.010
- VernacularTitle: 敲除组蛋白甲基转移酶PR结构域锌指蛋白9抑制牙周膜干细胞成骨向分化
- Author:
Jianpeng ZHANG
1
;
Haoqing YANG
2
;
Yan YAN
1
;
Benxiang HOU
1
Author Information
1. Department of Endodontics, School of Stomatology, Capital Medical University, Beijing 100050, China
2. Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Institute of Dental Research, School of Stomatology, Capital Medical University, Beijing 100050, China
- Publication Type:Journal Article
- Keywords:
Methyltransferase;
Periodontal ligament;
Mesenchymal stem cells;
PR domain zinc finger protein 9;
Osteogenesis differentiation
- From:
Chinese Journal of Stomatology
2019;54(12):841-846
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of PR domain zinc finger protein 9 (PRDM9), one of the histone methylated transferases, on osteogenic differentiation ability of periodontal ligament mesenchymal stem cells (PDLSC).
Methods:PDLSC with PRDM9 gene knocked down by PRDM9 shRNA using recombinant lentiviral vector were allocated into the PRDM9sh group, and the transfected shRNA was as the control group. The gene expression efficiency was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Alkaline phosphatase activity (ALP), alizarin red staining, mineralization and osteocalcin, which belongs to osteogenic differentiation markers detected by RT-PCR and Western blotting to detect the osteogenic differentiation ability of stem cells from periodontal ligaments in vitro. In vivo, PRDM9sh and control group cells was transplanted into the dorsal dermal to explore the osteogenesis. The area percentage of new osteogenic tissue was calculated by image pro software and statistically analyzed.
Results:RT-PCR results showed that the relative expression of PRDM9 gene in PRDM9sh (0.460±0.017) was significantly lower than that in control group (1.000±0.107) (P<0.05). The results of ALP activity determined at 5 days postinduction in a significant decrease in PRDM9sh cells (0.762±0.063) compared with control group (1.225±0.058) (P<0.01). Alizarin red staining induced by osteogenesis at 2 weeks and 3 weeks showed that the staining of PRDM9sh was significantly lighter than that in control group. Quantitative calcium analysis results showed that the calcium ion concentration induced by osteogenesis at 2 weeks and 3 weeks [(0.071±0.004), (0.075±0.001)] in PRDM9sh was significantly lower than that in control group at 2 weeks and 3 weeks [(0.282±0.006), (0.485+0.004)] (P<0.01). RT-PCR results showed that the relative expression of osteocalcin mRNA in PRDM9sh (1.059±0.148) was significantly lower than that in control group at 2 weeks (2.542±0.190) (P<0.01). Western blotting results showed that osteocalcin expression in PRDM9sh was significantly lower than that in control group at 1 and 2 weeks after osteogenesis induction. Animal transplantation experiments results indicated that PRDM9 significantly inhibited the osteogenesis of PDLSC in vivo, and the proportion of osteogenic area calculated showed that the osteogenic capacity of PRDM9sh [(3.8±2.41)%] was significantly lower than that in control group [(24.54±7.06)%](P<0.05).
Conclusions:Depletion of PRDM9 repressed the osteogenic differentiation of stem cells from periodontal ligament in vitro and in vivo.
