Wu Yun, Ou Yanjing, Liao Chufang, Liang Shanshan, Wang Yining
The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and The Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University Wuhan, PR China.
Department of Prosthodontics, Hospital of Stomatology, Wuhan University Wuhan, PR China.
Am J Transl Res. 2019 Jun 15;11(6):3398-3411. eCollection 2019.
Mechanical tension force directs the lineage commitment of periodontal ligament cells (PDLCs) to osteogenesis; however, the underlying mechanisms, especially those at the post-transcriptional level, remain unclear. In the present study, we developed an in vitro force-loading model for PDLCs. Then, high-throughput sequencing was used to identify the expression profile of microRNAs (miRNAs) for stretched PDLCs. The candidate target genes of differentially expressed miRNAs were predicted by bioinformatics analysis. A total of 47 miRNAs were found to be differentially expressed in stretched and non-stretched PDLCs; of these, 31 were upregulated and 16 were downregulated. Further, 9 osteogenesis-related miRNAs (miR-221-3p, miR-138-5p, miR-132-3p, miR-218-5p, miR-133a-3p, miR-145-3p, miR-143-5p, miR-486-3p, and miR-21-3p) were validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis were then carried out to reveal the potential functions of predicted target genes. Among the top 20 enriched pathways, the Hippo signaling pathway was selected for further functional analysis. Several important components of the Hippo signaling pathway, including YAP1, WWTR1, TEAD2, CTGF, DVL2, GDF5, GLI2, LIMD1, WTIP, LATS1, and TEAD1, were predicted to be target genes of differentially expressed miRNAs and were determined to be upregulated in stretched PDLCs. Among them, YAP1, WWTR1, TEAD2, CTGF, DVL2, and GDF5 were positive regulators of osteogenesis. These findings may provide a reliable reference for future studies to elucidate the biological mechanisms of orthodontic tooth movement (OTM).
机械张力引导牙周膜细胞（PDLCs）向成骨方向分化；然而，其潜在机制，尤其是转录后水平的机制仍不清楚。在本研究中，我们建立了一种用于PDLCs的体外力加载模型。然后，利用高通量测序确定拉伸后PDLCs中微小RNA（miRNAs）的表达谱。通过生物信息学分析预测差异表达miRNAs的候选靶基因。共发现47种miRNAs在拉伸和未拉伸的PDLCs中差异表达；其中，31种上调，16种下调。此外，通过定量逆转录-聚合酶链反应（RT-qPCR）验证了9种与成骨相关的miRNAs（miR-221-3p、miR-138-5p、miR-132-3p、miR-218-5p、miR-133a-3p、miR-145-3p、miR-143-5p、miR-486-3p和miR-21-3p）。随后进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路分析，以揭示预测靶基因的潜在功能。在前20个富集通路中，选择Hippo信号通路进行进一步功能分析。Hippo信号通路的几个重要组成部分，包括YAP1、WWTR1、TEAD2、CTGF、DVL2、GDF5、GLI2、LIMD1、WTIP、LATS1和TEAD1，被预测为差异表达miRNAs的靶基因，并被确定在拉伸的PDLCs中上调。其中，YAP1、WWTR1、TEAD2、CTGF、DVL2和GDF5是成骨的正调节因子。这些发现可能为未来阐明正畸牙移动（OTM）生物学机制提供可靠参考。
