NELL-1 regulates the P1 promoter of RUNX2 to promote osteogenic differentiation

doi:10.3877/cma.j.issn.1674-1366.2017.04.002

Abstract

Abstract:

Objective

To investigate regulation mechanism between the osteoinductive factor NELL-1 and the osteogenic transcription factor RUNX2.

Methods

The EGFP lentivirus vector with the P1 promoter of RUNX2 were constructed, obtained and transfected the M2-10B4 cells. The activity of the P1 Promoter of RUNX2 after the treatment of different doses of BMP-2 protein or NELL-1 protein were determined by EGFP gene expression analysis using flow cytometry. Alizarin Red Staining Quantification Assay evaluated the calcium nodules in three groups during the osteoblastic differentiation of mouse MSCs. The bone formation-related genes OPN, OCN, ALP and RUNX2 expression were detected by real-time PCR. Data were analyzed using One-Way ANOVA procedure.

Results

The 800 ng/ml or 1600 ng/ml NELL-1-induced increase of P1 promoter of RUNX2 by 82.09% or 92.36% was statistically significant, compared with control (F_N800= 26.979, P_N800<0.05; F_N1600= 28.410, P_N1600<0.01) . Alizarin Red Staining Quantification Assay indicated that 800 ng/ml NELL-1 group in calcium compounds was dramatically reduced by 73.41%, compared with control (F_N800= 31.179, P_N800<0.01) ; Real-time PCR detected that NELL-1 can promote the expression of OPN, OCN, ALP and RUNX2 during the differentiation process in mouse mesenchymal stem cells.

Conclusion

NELL-1 enhance the osteoblastic differentiation of mouse MSCs by promote the expression of RUNX2 by its P1 promoter.

Key words: Bone marrow, Stromal cells, Mice, NELL-1 gene, Osteogenic differentiation, Lentivirusvector

Hong Zhang, Yu Yao, Jiadong Sun, Xiaonan Yu, Zhiguang Zhang. NELL-1 regulates the P1 promoter of RUNX2 to promote osteogenic differentiation[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(04): 197-203.

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Figures/Tables 6

References 21

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成骨相关基因	引物序列
RUNX2	上游：5′-TTCCCCTTCCCCTCCCTTTATC-3′
?	下游：5′-CTAAGCCCAGGGAAGTTTGTACA-3′
ALP	上游：5′-GACGGTGAACGGGAGAAC-3′
?	下游：5′-CTCAGAACAGGGTGCGTAG-3′
OCN	上游：5′-TAAGGTGGTGAATAGACTCCG-3′
?	下游：5′-GTGCCGTCCATACTTTCG-3′
OPN	上游：5′-TGAAGCCTGACCCATCTC-3′
?	下游：5′-GGTCTTCCCGTTGCTGTC-3′

成骨相关基因	引物序列
RUNX2	上游：5′-TTCCCCTTCCCCTCCCTTTATC-3′
?	下游：5′-CTAAGCCCAGGGAAGTTTGTACA-3′
ALP	上游：5′-GACGGTGAACGGGAGAAC-3′
?	下游：5′-CTCAGAACAGGGTGCGTAG-3′
OCN	上游：5′-TAAGGTGGTGAATAGACTCCG-3′
?	下游：5′-GTGCCGTCCATACTTTCG-3′
OPN	上游：5′-TGAAGCCTGACCCATCTC-3′
?	下游：5′-GGTCTTCCCGTTGCTGTC-3′

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