Mechanism of HDAC3-Targeted Regulation of GATA-2 Affecting Osteogenic Differentiation of Dental Pulp Stem Cells
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摘要:
目的 探讨HDAC3/GATA-2分子轴在人牙髓干细胞（human dental pulp stem cells，hDPSCs）成骨分化中的作用机制。 方法 hDPSCs细胞系通过骨诱导培养基（osteogenic induction medium，OM）培养，构建oe-NC，oe-HDAC3和oe-GATA-2质粒，并转染至细胞中，转染效率通过RT-qPCR和Western blot进行评估。采用碱性磷酸酶（alkaline phosphatase，ALP）染色和茜素红S（alizarin red S，ARS）染色评估ALP活性以及细胞的矿化节点。RT-qPCR和Western Blot检测成骨关键指标COL1A1、ALP、BMP2和RUNX2的表达水平。GST-pull down和免疫共沉淀（co-immunoprecipitation，CO-IP）验证HDAC3与GATA-2的相互作用，免疫荧光（immunofluorescence，IF）染色检测细胞表达。 结果 HDAC3在hDPSCs成骨分化过程中显著降低（P < 0.0001 ），过表达HDAC3显著减弱ALP染色和活性（P =0.0001 ），以及减少了hDPSCs细胞矿化结节的产生。COL1A1（P =0.0029 ）、ALP（P =0.0001 ）、BMP2（P =0.0001 ）和RUNX2（P =0.0007 ）的表达水平在过表达HDAC3后被抑制。HDAC3与GATA-2相互作用，与过表达HDAC3降低了细胞中GATA-2的表达（P =0.0028 ）。共转染oe-GATA-2后逆转了过表达HDAC3的作用，增加了ALP染色和活性（P =0.0043 ）和细胞中的矿化结节。oe-HDAC3+ oe-GATA-2组中COL1A1（P =0.0001 ）、ALP（P =0.0423 ）、BMP2（P <0.0001 ）和RUNX2（P =0.0005 ）的表达水平进一步升高。结论 HDAC3通过靶向负调控GATA-2的表达，抑制了hDPSCs的成骨分化。 Abstract:Objective To investigate the mechanism of the HDAC3/GATA-2 molecular axis in osteogenic differentiation of human dental pulp stem cells （hDPSCs）. Methods The hDPSCs cell line was cultured in osteogenic induction medium （OM）. Plasmids of oe-NC, oe-HDAC3 and oe-GATA-2 were constructed and transfected into the cells. Transfection efficiency was assessed by RT-qPCR and Western blot. Alkaline phosphatase （ALP） staining and alizarin red S （ARS） staining were used to evaluate ALP activity and mineralization nodules in cells. RT-qPCR and Western Blot were used to detect the expression levels of osteogenic key indicators COL1A1, ALP, BMP2, and RUNX2. GST-pull down and Co-Immunoprecipitation （CO-IP） were used to validate the interaction of HDAC3 and GATA-2 interaction, and immunofluorescence （IF） staining was performed to detect cellular expression. Results HDAC3 expression was significantly reduced during osteogenic differentiation of hDPSCs （P < 0.0001 ）. Overexpression of HDAC3 significantly attenuated ALP staining and activity （P =0.0001 ）, as well as reduced the production of mineralized nodules in hDPSCs cells. The expression levels of COL1A1 （P =0.0029 ）, ALP （P =0.0001 ）, BMP2 （P =0.0001 ） and RUNX2 （P =0.0007 ） were inhibited after overexpression of HDAC3. HDAC3 interacted with GATA-2, and overexpression of HDAC3 decreased the expression of GATA-2 in cells （P =0.0028 ）. Co-transfection of oe-GATA-2 reversed the inhibitory effects of HDAC3 overexpression, increasing ALP staining and activity （P =0.0043 ） and mineralization nodules in cells. In the oe-HDAC3 + oe-GATA-2 group, the expression levels of COL1A1 （P =0.0001 ）, ALP （P =0.0423 ）, BMP2 （P <0.0001 ）, and RUNX2 （P =0.0005 ） were further elevated.Conclusion HDAC3 inhibited the osteogenic differentiation of hDPSCs by targeting and negatively regulating the expression of GATA-2. -
Key words:
- Human dental pulp stem cells /
- Osteogenic differentiation /
- HDAC3 /
- GATA-2
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图 1 HDAC3在hDPSCs成骨分化过程中的低表达。
A: RT-qPCR；B：Western blot检测COL1A1、ALP、BMP2、RUNX2和HDAC3的mRNA和蛋白表达；与0 d组相比，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 1. Low expression of HDAC3 during osteogenic differentiation of hDPSCs.
图 2 过表达HDAC3抑制hDPSCs成骨分化
A：RT-qPCR；B：Western blot检测转染效率；C：ALP染色（Scale bar=100 μm）和ARS染色（Scale bar=200 μm）；D：试剂盒检测ALP活性；E：RT-qPCR和F：Western blot检测COL1A1、ALP、BMP2和RUNX2的mRNA和蛋白表达；与NC组相比，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 2. Overexpression of HDAC3 inhibits osteogenic differentiation of hDPSCs
图 3 HDAC3靶向抑制GATA-2的表达
A：HDAC3靶基因预测；B：GST - pulled down验证HDAC3与GATA-2的结合；C：CO-IP验证HDAC3与GATA-2的相互作用；D：IF检测细胞共定位（Scale bar=20 μm）；E：RT-qPCR和F：Western blot检测GATA-2的mRNA和蛋白表达；与NC组相比，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 3. HDAC3 targets and inhibits GATA-2 expression
图 4 HDAC3通过下调GATA-2抑制hDPSCs成骨分化
A：RT-qPCR；B：Western blot检测转染效率；C：ALP染色（Scale bar=100 μm）和ARS染色（Scale bar=200 μm）；D：试剂盒检测ALP活性；E：RT-qPCR和F：Western blot检测COL1A1、ALP、BMP2和RUNX2的mRNA和蛋白表达；与oe-NC组相比，*P < 0.05，**P < 0.01，***P < 0.001；与oe-HDAC3组相比，#P < 0.05，##P < 0.01，###P < 0.001。
Figure 4. HDAC3 inhibits hDPSCs osteogenic differentiation through downregulation of GATA-2
表 1 PCR引物序列
Table 1. PCR primer sequences
基因 引物 序列（5’-3’） HDAC3 正向 5’-GGTGGTTATACTGTCCGAAAT-3’ 反向 5’-AGGCTGAAGTCCCTGCTC-3’ COL1A1 正向 5’-AAGGTGTTGTGCGATGACG-3’ 反向 5’-ACCACGAGGACCAGAGGGAC-3’ ALP 正向 5’-GGCTGGACGGGAAGAATC-3’ 反向 5’-GCCTCCGAAGGAGAAGACG-3’ BMP2 正向 5’-AACCTGCAACAGCCAACT-3’ 反向 5’-GGGAGCCACAATCCAGTC-3’ RUNX2 正向 5’-TGGGCTTCCTGCCATCAC-3’ 反向 5’-CAGCGTCAACACCATCATTC-3’ GATA-2 正向 5’-CTCCTGACCCTAGCACCACG-3’ 反向 5’-GGTTCTGCCCATTCATCTTGT-3’ GAPDH 正向 5’-TGACCACAGTCCATGCCATCAC-3’ 反向 5’-CGCCTGCTTCACCACCTTCTT-3’ 
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