Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, People's Republic of China.
College of Stomatology, Southern Medical University, Guangzhou, 510515, Guangdong, People's Republic of China.
Stem Cell Res Ther. 2020 Mar 13;11(1):114. doi: 10.1186/s13287-020-01622-w.
Long noncoding RNAs (lncRNAs) play an important role in the multiple differentiations of mesenchymal stem cells (MSCs). However, few studies have focused on the regulatory mechanism of lncRNAs in the odontogenic differentiation of human dental pulp stem cells (hDPSCs).
hDPSCs were induced to differentiate into odontoblasts in vitro, and the expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in differentiated and undifferentiated cells were obtained by microarray. Bioinformatics analyses including Gene Ontology (GO) analysis, pathway analysis, and binding site prediction were performed for functional annotation of lncRNA. miRNA/odontogenesis-related gene networks and lncRNA-associated ceRNA networks were constructed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the expression of selected genes. RNA fluorescence in situ hybridization (FISH), qRT-PCR, and western blot analysis were used to explore the location and function of lncRNA-G043225. Dual-luciferase reporter assay was performed to confirm the binding sites of miR-588 with G043225 and Fibrillin 1 (FBN1).
We identified 132 lncRNAs, 114 miRNAs, and 172 mRNAs were differentially expressed. GO analysis demonstrated that regulation of the neurogenic locus notch homolog (Notch), Wnt, and epidermal growth factor receptor (ERBB) signaling pathways and activation of mitogen-activated protein kinase (MAPK) activity were related to odontogenic differentiation. Pathway analysis indicated that the most significant pathway was the forkhead box O (FoxO) signaling pathway, which is related to odontogenic differentiation. Two odontogenesis-related gene-centered lncRNA-associated ceRNA networks were successfully constructed. The qRT-PCR validation results were consistent with the microarray analysis. G043225 mainly locating in cytoplasm was proved to promote the odontogenic differentiation of hDPSCs via miR-588 and FBN1.
This is the first study revealing lncRNA-associated ceRNA network during odontogenic differentiation of hDPSCs using microarray, and it could provide clues to explore the mechanism of action at the RNA-RNA level as well as novel treatments for dentin regeneration based on stem cells.
长非编码 RNA（lncRNA）在间充质干细胞（MSC）的多种分化中发挥重要作用。然而，很少有研究关注 lncRNA 在人牙髓干细胞（hDPSC）成牙分化中的调控机制。
体外诱导 hDPSC 向成牙本质细胞分化，通过微阵列获得分化和未分化细胞中 lncRNA、微小 RNA（miRNA）和信使 RNA（mRNA）的表达谱。进行基因本体（GO）分析、通路分析和结合位点预测等生物信息学分析，对 lncRNA 进行功能注释。构建 miRNA/牙发生相关基因网络和 lncRNA 相关 ceRNA 网络。采用定量逆转录聚合酶链反应（qRT-PCR）验证选定基因的表达。采用 RNA 荧光原位杂交（FISH）、qRT-PCR 和 Western blot 分析探讨 lncRNA-G043225 的位置和功能。双荧光素酶报告基因检测证实 miR-588 与 G043225 和原纤维蛋白 1（FBN1）的结合位点。
鉴定出 132 个 lncRNA、114 个 miRNA 和 172 个 mRNA 差异表达。GO 分析表明，神经源性基因座 Notch 同源物（Notch）、Wnt 和表皮生长因子受体（ERBB）信号通路的调节以及丝裂原激活蛋白激酶（MAPK）活性的激活与牙发生分化有关。通路分析表明，最显著的通路是叉头框 O（FoxO）信号通路，该通路与牙发生分化有关。成功构建了两个以牙发生相关基因为中心的 lncRNA 相关 ceRNA 网络。qRT-PCR 验证结果与微阵列分析一致。G043225 主要位于细胞质中，通过 miR-588 和 FBN1 促进 hDPSC 的牙向分化。
这是首次使用微阵列研究 hDPSC 牙向分化过程中 lncRNA 相关 ceRNA 网络，可为探索 RNA-RNA 水平作用机制以及基于干细胞的牙本质再生新疗法提供线索。
