Dataset Information

High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells.

ABSTRACT: 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).

SUBMITTER: Wu Y

PROVIDER: S-EPMC6614645 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells.

Wu Yun Y Ou Yanjing Y Liao Chufang C Liang Shanshan S Wang Yining Y

American journal of translational research 20190615 6

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 bioinfo ...[more]

PMID: 31312353

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Project description:The prevalence of obesity has significantly increased worldwide. Therefore, this study aimed to evaluate the influence of obesity in the proteomic profile of periodontal ligament (PDL) tissues of rat first maxillary molars submitted to orthodontic tooth movement. The study was approved by the Ethical Committee on Animal Experimentation of the Sao Paulo State University, School of Dentistry at Araraquara, Brazil (protocol number 16/2015) and followed the Animal Research: Reporting of In Vivo Experiments guidelines. Therefore, for this study, we used ten male adult Holtzman rats (weight average – 300 g, approximately 8 weeks old). Animals were randomly assigned to one of the following experimental groups: 1 – M group: animals received a standard diet and 75 days after that, the first maxillary molar was randomly selected to be submitted to OTM for 15 days (n=5), and 2 – OM group: animals received a high-fat diet to induce obesity, and 75 days after that the first maxillary molar was randomly selected to be subjected to OTM for 15 days (n=5). All animals were kept under controlled temperature (22-25 °C), receiving water ad libitum with a 12-hour light/dark cycle. To induce obesity, animals of the OM group received a high-fat diet. In contrast to the standard diet (Labina/Purina, Ribeirão Preto, Brazil) administered to the M group. The high-fat diet contained around 3.82 kcal/g and was composed of 27.5 % of standard rat chow plus 25 % of peanuts, 25 % of milk chocolate, and 12.5 % sweet biscuits and contained 20 g of protein, 48 g of carbohydrate, 20 g of total fat, and 4 g of fiber per 100 g of diet. This feed was daily replaced and had started 75 days before baseline (M initiation). The procedure to induce OTM was by a closed coil nickel-titanium spring (Sentalloy, GAC Central Slip, New York, EUA) connected the maxillary first molar and central incisors with a steel wire (CrNi, 0.20 mm, 55.01.208; Morelli, Sorocaba, Brazi). Fifteen days after OTM, animals were euthanized by anesthetic overdose.

Project description:Periodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs.The expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining.We observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3' untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis.Our results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.

Dataset Information

High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells.

Publications

High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells.

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