Department of Stomatology, Beijing Children's Hospital, National Center for Children's Health, Capital Medical University, Beijing, China.
Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing, China.
Orthod Craniofac Res. 2023 Nov;26(4):667-678. doi: 10.1111/ocr.12668. Epub 2023 May 2.
Stimulation of cementogenesis is essential to cementum regeneration and root restoration. Long non-coding RNAs (lncRNAs) participate in the regulatory networks of periodontal regeneration processes. We identified and analysed differentially expressed lncRNAs, miRNAs and mRNAs associated with cementogenic differentiation of cementoblasts.
OCCM-30 immortalized mouse cementoblast cells were induced in cementogenic medium for 7 and 14 days. Total RNA was extracted and subjected to RNA sequencing to screen for differentially expressed lncRNAs, miRNAs and mRNAs. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of RNAs. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to clarify the potential functions of differentially expressed genes in biological processes and pathways. lncRNA-miRNA-mRNA networks were constructed based on correlation and algorithmic analyses.
In all, 461 lncRNAs, 89 miRNAs and 2157 mRNAs showed differential expression in OCCM-30 cells after cementoblast differentiation. At day 7, upregulation of 248 lncRNAs, 30 miRNAs and 905 mRNAs was observed, along with downregulation of 127 lncRNAs, 34 miRNAs and 960 mRNAs. At day 14, 197 lncRNAs, 13 miRNAs and 847 mRNAs were upregulated, while 74 lncRNAs, 12 miRNAs and 760 mRNAs were downregulated. The results of qRT-PCR showed that four candidate lncRNAs, H19, Gdap10, Foxo6os and Ipw, were significantly upregulated after 7 and 14 days of cementogenic induction. The lncRNA-miRNA-mRNA network illustrated a possible competitive endogenous RNA regulatory mechanism. GO analysis showed that consistently differentially expressed mRNAs were involved in blood vessel morphogenesis, cell-substrate adhesion, cell adhesion, ossification and extracellular matrix organization. KEGG analysis indicated that extracellular matrix-receptor interaction, focal adhesion, and the PI3K-Akt, Rap1, mitogen-activated protein kinase, and Ras signalling pathways varied significantly during cementogenesis.
The expressions of lncRNA, miRNA and mRNA were significantly altered in cementoblasts after cementogenesis. This study highlighted the effect of lncRNAs in the process of cementogenesis and revealed their potential for the discovery of novel biomarkers and therapeutic targets for cementum regeneration.
促进牙骨质发生对于牙骨质再生和牙根修复至关重要。长链非编码 RNA（lncRNA）参与牙周组织再生过程的调控网络。我们鉴定并分析了与成牙骨质细胞成牙骨质分化相关的差异表达的 lncRNA、miRNA 和 mRNA。
将永生化的小鼠成牙骨质细胞（OCCM-30）在成牙骨质培养基中诱导培养 7 天和 14 天。提取总 RNA 并进行 RNA 测序，以筛选差异表达的 lncRNA、miRNA 和 mRNA。采用实时定量聚合酶链反应（qRT-PCR）测定 RNA 的表达水平。通过基因本体论（GO）术语和京都基因与基因组百科全书（KEGG）通路分析，阐明差异表达基因在生物学过程和通路中的潜在功能。基于相关性和算法分析构建 lncRNA-miRNA-mRNA 网络。
在成牙骨质细胞分化后，OCCM-30 细胞中共有 461 个 lncRNA、89 个 miRNA 和 2157 个 mRNA 显示差异表达。在第 7 天，观察到 248 个 lncRNA、30 个 miRNA 和 905 个 mRNA 上调，同时有 127 个 lncRNA、34 个 miRNA 和 960 个 mRNA 下调。在第 14 天，有 197 个 lncRNA、13 个 miRNA 和 847 个 mRNA 上调，同时有 74 个 lncRNA、12 个 miRNA 和 760 个 mRNA 下调。qRT-PCR 结果显示，在成牙骨质诱导培养 7 天和 14 天后，有 4 个候选 lncRNA（H19、Gdap10、Foxo6os 和 Ipw）显著上调。lncRNA-miRNA-mRNA 网络表明存在一种可能的竞争性内源 RNA 调控机制。GO 分析表明，一致差异表达的 mRNA 参与血管形态发生、细胞-基质黏附、细胞黏附、骨化和细胞外基质组织。KEGG 分析表明，在成牙骨质过程中，细胞外基质-受体相互作用、焦点黏附、PI3K-Akt、Rap1、丝裂原活化蛋白激酶和 Ras 信号通路显著改变。
成牙骨质细胞成牙骨质分化后，lncRNA、miRNA 和 mRNA 的表达明显改变。本研究强调了 lncRNA 在成牙骨质过程中的作用，并揭示了它们在牙骨质再生中发现新的生物标志物和治疗靶点的潜力。
