长链非编码 RNA H19 通过调控 miR-140-5p 和 BMP-2/FGF9 促进人牙髓干细胞的成牙本质分化。
LncRNA H19 promotes odontoblastic differentiation of human dental pulp stem cells by regulating miR-140-5p and BMP-2/FGF9.
机构信息
Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatology Hospital of Guangzhou Medical University, Huangsha Avenue 39, Guangzhou, 510000, People's Republic of China.
State Key Laboratory of Respiratory Disease, Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou, 511436, People's Republic of China.
出版信息
Stem Cell Res Ther. 2020 May 27;11(1):202. doi: 10.1186/s13287-020-01698-4.
BACKGROUND
Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs).
METHODS
We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified as a critical factor by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot, and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs.
RESULTS
The expression of H19 was significantly upregulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas downregulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation.
CONCLUSION
Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.
背景
越来越多的证据表明，长非编码 RNA（lncRNA）在生物矿化中发挥着关键作用。成牙本质细胞分化是牙本质形成的关键过程，受复杂的信号网络调控。本研究旨在探讨 lncRNA-H19（H19）在调控人牙髓干细胞（hDPSCs）成牙本质分化中的生物学作用和调控机制。
方法
我们进行了 lncRNA 微阵列分析，以揭示参与成牙本质分化的 lncRNA 的表达模式。通过 qRT-PCR 鉴定并验证 H19 是一个关键因子。通过 gain-和 loss-of-function 研究，在体外和体内研究 H19 调节 hDPSCs 成牙本质分化的生物学作用。通过 qRT-PCR、Western blot 和茜素红 S 染色评估成牙本质分化。生物信息学分析表明，H19 可以直接与 miR-140-5p 相互作用，这通过荧光素酶报告基因实验进一步验证。在 hDPSCs 中转染 miR-140-5p 过表达后，确定成牙本质分化。此外，还研究了 miR-140-5p 的潜在靶基因，并验证了 BMP-2 和 FGF9 在 hDPSCs 中的生物学功能。进行共转染实验以验证 miR-140-5p 是否参与 hDPSCs 中的 H19 介导的成牙本质分化。
结果
在 hDPSCs 成牙本质分化过程中，H19 的表达明显上调。H19 的过表达在体外和体内均刺激成牙本质分化，而 H19 的下调则呈现相反的效果。H19 直接与 miR-140-5p 结合，过表达 miR-140-5p 抑制 hDPSCs 的成牙本质分化。H19 作为 miR-140-5p 的海绵，调节 BMP-2 和 FGF9 的表达。过表达 H19 消除了 miR-140-5p 对成牙本质分化的抑制作用。
结论
我们的数据表明，H19 通过 miR-140-5p/BMP-2/FGF9 轴在 hDPSCs 的成牙本质分化中发挥正向调节作用，提示 H19 可能是牙发生的刺激调节因子。
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