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.
American journal of translational research
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).
Integrated miRNA and mRNA expression profiling of tension force-induced bone formation in periodontal ligament cells.
Chang Maolin,Lin Heng,Luo Meng,Wang Jie,Han Guangli
In vitro cellular & developmental biology. Animal
Tension force-induced bone formation is a complex biological process altered by various factors, for example miRNAs and gene regulatory network. However, we know little about critical gene regulators and their functional consequences on this complex process. The aim of this study was to determine the integrated relation between microRNA and mRNA expression in tension force-induced bone formation in periodontal ligament cells by a system biological approach. We identified 818 mRNAs and 32 miRNAs differentially expressed between cyclic tension force-stimulated human periodontal ligament cells and control cells by microarrays. By using miRNA/mRNA network analysis, protein-protein interactions network analysis, and hub analysis, we found that miR-195-5p, miR-424-5p, miR-1297, miR-3607-5p, miR-145-5p, miR-4328, and miR-224-5p were core microRNAs of tension force-induced bone formation. WDR33, HSPH1, ERBB3, RIF1, IKBKB, CREB1, FGF2, and PAG1 were identified as hubs of the PPI network, suggesting the biological significance in this process. The miRNA expression was further examined in human PDLC and animal samples by using quantitative real-time PCR. Thus, we proposed a model of tension force-induced bone formation which is co-regulated through integration of the miRNA and mRNA. This study illustrated the benefits of system biological approaches in the analysis of tension force-induced bone formation as a complex biological process. We used public information and our experimental data to do comprehensive analysis and revealed the coordination transcriptional control of miRNAs of tension force-induced bone formation.
10.1007/s11626-015-9892-0
Analysis of lncRNAs-miRNAs-mRNAs networks in periodontal ligament stem cells under mechanical force.
Wang Hong,Feng Cheng,Li Mengying,Zhang Zijie,Liu Jiani,Wei Fulan
Oral diseases
OBJECTIVES:Our study aims to analyze the expression profiles of long non-coding RNAs (lncRNAs) and investigate the potential regulatory networks among lncRNAs, microRNAs (miRNAs), and mRNAs in periodontal ligament stem cells (PDLSCs) under mechanical force (MF). MATERIALS AND METHODS:PDLSCs were isolated from human periodontal ligament tissues and identified by flow cytometry analysis. Multidirectional differentiation potential of PDLSCs was obtained by osteogenic and adipogenic induction. High-throughput RNA sequencing was used to identify the expression patterns of lncRNAs and mRNAs in PDLSCs under MF. MF-responsive miRNAs were obtained from the previous microarray data. LncRNAs-miRNAs-mRNAs networks were constructed by Cytoscape. RESULTS:PDLSCs cultured from the periodontal ligament tissues were positive for STRO-1, CD146 and negative for CD45, CD34. Alizarin red staining and Oil Red O staining showed that PDLSCs had the ability of osteogenic and adipogenic differentiation. Then, a total of 1,339 and 1,426 differentially expressed lncRNAs and mRNAs were identified, respectively, in PDLSCs under MF. Based on the previous miRNA microarray analysis, the potential interaction networks of lncRNAs-miRNAs-mRNAs were constructed. It was found that lncRNAs and mRNAs could competitively interact with the same miRNA. CONCLUSIONS:LncRNAs-miRNAs-mRNAs networks were involved in PDLSCs under MF, which might provide a novel mechanism in the regulation of clinical orthodontic tooth movement process.
10.1111/odi.13530
Identification and characterization of circular RNAs involved in mechanical force-induced periodontal ligament stem cells.
Wang Hong,Feng Cheng,Jin Ye,Tan Wanye,Wei Fulan
Journal of cellular physiology
Circular RNAs (circRNAs) play critical roles in signal transduction during cell proliferation, differentiation, and apoptosis in a posttranscriptional manner. Recently, circRNAs have been proved to be a large class of animal RNAs with regulatory potency. However, whether circRNAs can respond to mechanical force (MF) and impact on human periodontal ligament stem cells (PDLSCs) and the orthodontic tooth movement (OTM) process remain unknown. Here, we investigated the circRNAs expression patterns in PDLSCs induced by MF and found that circRNAs were responsive to the MF in PDLSCs. Through the valid reads' distribution analysis, we found that the majority of reads in both the control PDLSCs and the MF-induced PDLSCs were distributed in exons. Then we analyzed Gene Ontology terms of genes that overlap with or are neighbors of the stress-responsive circRNAs and found unique enrichment patterns in biological processes, molecular function, and cellular component of PDLSCs. Next, we predicted the possible functions of circRNAs through circRNAs-miRNAs networks. We found that one circRNA may regulate one or several miRNA/miRNAs and one miRNA may interact with one or multiple circRNA/circRNAs. Importantly, a number of circRNAs were predicted to directly or indirectly regulate miRNAs-mediated osteogenic differentiation in mesenchymal stem cells. For instance, circRNA3140 was highly and widely associated with microRNA-21, which plays a critical role in MF-induced osteogenic differentiation of PDLSCs. Taken together, these findings reveal a previously unrecognized mechanism that MF can induce the expression changes of circRNAs in PDLSCs, which may modulate the OTM process and the alveolar bone remodeling.
10.1002/jcp.27686
Transcriptome Reveals Cathepsin K in Periodontal Ligament Differentiation.
Yamada S,Ozaki N,Tsushima K,Yamaba S,Fujihara C,Awata T,Sakashita H,Kajikawa T,Kitagaki J,Yamashita M,Yanagita M,Murakami S
Journal of dental research
Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell differentiation into hard tissue-forming cells.
10.1177/0022034516645796
The long non-coding RNA landscape of periodontal ligament stem cells subjected to compressive force.
Huang Yiping,Zhang Yingying,Li Xiaobei,Liu Hao,Yang Qiaolin,Jia Lingfei,Zheng Yunfei,Li Weiran
European journal of orthodontics
OBJECTIVE:The role of long non-coding ribonucleic acids (lncRNAs) during orthodontic tooth movement remains unclear. We explored the lncRNA landscape of periodontal ligament stem cells (PDLSCs) subjected to compressive force. MATERIALS AND METHODS:PDLSCs were subjected to static compressive stress (2 g/cm2) for 12 hours. Total RNA was then extracted and sequenced to measure changes in lncRNA and messenger RNA (mRNA) expression levels. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the expression levels of certain lncRNAs. Differential expression analysis as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were also performed. RESULTS:In total, 90 lncRNAs and 519 mRNAs were differentially expressed in PDLSCs under compressive stress. Of the lncRNAs, 72 were upregulated and 18 downregulated. The levels of eight lncRNAs of interest (FER1L4, HIF1A-AS2, MIAT, NEAT1, ADAMTS9-AS2, LUCAT1, MIR31HG, and DHFRP1) were measured via qRT-PCR, and the results were found to be consistent with those of RNA sequencing. GO and KEGG pathway analyses showed that a wide range of biological functions were expressed during compressive loading; most differentially expressed genes were involved in extracellular matrix organization, collagen fibril organization, and the cellular response to hypoxia. CONCLUSIONS:The lncRNA expression profile was significantly altered in PDLSCs subjected to compressive stress. These findings expand our understanding of molecular regulation in the mechanoresponse of PDLSCs.
10.1093/ejo/cjy057
Long noncoding RNA expression profile of mouse cementoblasts under compressive force.
Liu Hao,Huang Yiping,Zhang Yingying,Han Yineng,Zhang Yixin,Jia Lingfei,Zheng Yunfei,Li Weiran
The Angle orthodontist
OBJECTIVES:To investigate the long noncoding RNA (lncRNA) expression profile of cementoblasts under compressive force. MATERIALS AND METHODS:Mouse cementoblasts were exposed to compression (1.5 g/cm) for 8 hours. RNA sequencing (RNA-seq) was performed to compare the transcriptomes of the compressed and control cells. Quantitative real-time polymerase chain reaction qRT-PCR) was used to validate five of the differentially expressed lncRNAs of interest. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were also performed. RESULTS:A total of 70 lncRNAs and 521 mRNAs were differentially regulated in cementoblasts subjected to compressive loading. Among the differentially expressed lncRNAs, 57 were upregulated and 13 downregulated. The expression levels of the five selected lncRNAs (Prkcz2, Hklos, Trp53cor1, Gdap10, and Ak312-ps) were validated by qRT-PCR and consistent with the RNA-seq results. GO functional annotation demonstrated upregulation of genes associated with cellular response to hypoxia and apoptotic processes during compressive loading. KEGG analysis identified the crucial pathways involving the hypoxia-inducing factor-1α, forkhead box O, and mammalian target of rapamycin signaling pathways. CONCLUSIONS:Mechanical compression changes the lncRNA expression profile of cementoblasts, providing important references for further investigation into the role and regulation of lncRNAs in compressed cementoblasts and root resorption during orthodontic treatment.
10.2319/061118-438.1
Immunorthodontics: in vivo gene expression of orthodontic tooth movement.
Scientific reports
Orthodontic tooth movement (OTM) is a "sterile" inflammatory process. The present study aimed to reveal the underlying biological mechanisms, by studying the force associated-gene expression changes, in a time-dependent manner. Ni-Ti springs were set to move the upper 1-molar in C57BL/6 mice. OTM was measured by μCT. Total-RNA was extracted from tissue blocks at 1,3,7 and 14-days post force application, and from two control groups: naïve and inactivated spring. Gene-expression profiles were generated by next-generation-RNA-sequencing. Gene Set Enrichment Analysis, K-means algorithm and Ingenuity pathway analysis were used for data interpretation. Genes of interest were validated with qRT-PCR. A total of 3075 differentially expressed genes (DEGs) were identified, with the greatest number at day 3. Two distinct clusters patterns were recognized: those in which DEGs peaked in the first days and declined thereafter (tissue degradation, phagocytosis, leukocyte extravasation, innate and adaptive immune system responses), and those in which DEGs were initially down-regulated and increased at day 14 (cell proliferation and migration, cytoskeletal rearrangement, tissue homeostasis, angiogenesis). The uncovering of novel innate and adaptive immune processes in OTM led us to propose a new term "Immunorthodontics". This genomic data can serve as a platform for OTM modulation future approaches.
10.1038/s41598-020-65089-8
Reference genes for valid gene expression studies on rat dental, periodontal and alveolar bone tissue by means of RT-qPCR with a focus on orthodontic tooth movement and periodontitis.
Kirschneck Christian,Proff Peter,Fanghänel Jochen,Wolf Michael,Roldán J Camilo,Römer Piero
Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft
OBJECTIVES:To obtain valid results in relative gene/mRNA-expression analyses by RT-qPCR, a careful selection of stable reference genes is required for normalization. Currently there is little information on reference gene stability in dental, periodontal and alveolar bone tissues of the rat, especially regarding orthodontic tooth movement and periodontitis. We therefore aimed to identify the best selection and number of reference genes under these experimental as well as physiological conditions. MATERIALS AND METHODS:In 7 male Fischer344-rats the upper left first and second molars were moved orthodontically for 2 weeks and in 7 more animals additionally subjected to an experimental periodontitis, whereas 7 animals were left untreated. Tissue samples of defined size containing both molars (without crowns) as well as the adjacent periodontal and alveolar bone tissue were retrieved and RNA extracted for RT-qPCR analyses. Nine candidate reference genes were evaluated and ranked according to their expression stability by 4 different algorithms (geNorm, NormFinder, BestKeeper, comparative ΔCq). RESULTS:PPIB/YWHAZ were the most stabile reference genes for the combined dental, periodontal and alveolar bone tissue of the rat overall, in untreated animals and rats with additional periodontitis, whereas PPIB/B2M performed best in orthodontically treated rats with YWHAZ ranking third. Gene-stability ranking differed considerably between investigated groups. A combination of two reference genes was found to be sufficient for normalization in all cases. CONCLUSIONS:The substantial differences in expression stability emphasize the need for valid reference genes, when aiming for meaningful results in relative gene expression analyses. Our results should enable researchers to optimize gene expression analysis in future studies by choosing the most suitable reference genes for normalization.
10.1016/j.aanat.2015.11.005
Effects of pre-applied orthodontic force on the regeneration of periodontal tissues in tooth replantation.
Korean journal of orthodontics
OBJECTIVE:This study aimed to investigate the effect of pre-applied orthodontic force on the regeneration of periodontal ligament (PDL) tissues and the underlying mechanisms in tooth replantation. METHODS:Orthodontic force (50 cN) was applied to the left maxillary first molars of 7-week-old male Sprague-Dawley rats (n = 32); the right maxillary first molars were left untreated to serve as the control group. After 7 days, the first molars on both sides were fully luxated and were immediately replanted in their original sockets. To verify the effects of the pre-applied orthodontic force, we assessed gene expression by using microarray analysis and real-time reverse transcription polymerase chain reaction (RT-PCR), cell proliferation by using proliferating cell nuclear antigen (PCNA) immunofluorescence staining, and morphological changes by using histological analysis. RESULTS:Application of orthodontic force for 7 days led to the proliferation of PDL tissues, as verified on microarray analysis and PCNA staining. Histological analysis after replantation revealed less root resorption, a better arrangement of PDL fibers, and earlier regeneration of periodontal tissues in the experimental group than in the control group. For the key genes involved in periodontal tissue remodeling, including , , , , , , and , quantitative RT-PCR confirmed that messenger RNA levels were higher at 1 or 2 weeks in the experimental group. CONCLUSIONS:These results suggest that the application of orthodontic force prior to tooth replantation enhanced the proliferation and activities of PDL cells and may lead to higher success rates with fewer complications.
10.4041/kjod.2019.49.5.299
LncRNA Nron regulates osteoclastogenesis during orthodontic bone resorption.
Zhang Ruilin,Li Junhui,Li Gongchen,Jin Fujun,Wang Zuolin,Yue Rui,Wang Yibin,Wang Xiaogang,Sun Yao
International journal of oral science
Activation of osteoclasts during orthodontic tooth treatment is a prerequisite for alveolar bone resorption and tooth movement. However, the key regulatory molecules involved in osteoclastogenesis during this process remain unclear. Long noncoding RNAs (lncRNAs) are a newly identified class of functional RNAs that regulate cellular processes, such as gene expression and translation regulation. Recently, lncRNAs have been reported to be involved in osteogenesis and bone formation. However, as the most abundant noncoding RNAs in vivo, the potential regulatory role of lncRNAs in osteoclast formation and bone resorption urgently needs to be clarified. We recently found that the lncRNA Nron (long noncoding RNA repressor of the nuclear factor of activated T cells) is highly expressed in osteoclast precursors. Nron is downregulated during osteoclastogenesis and bone ageing. To further determine whether Nron regulates osteoclast activity during orthodontic treatment, osteoclastic Nron transgenic (Nron cTG) and osteoclastic knockout (Nron CKO) mouse models were generated. When Nron was overexpressed, the orthodontic tooth movement rate was reduced. In addition, the number of osteoclasts decreased, and the activity of osteoclasts was inhibited. Mechanistically, Nron controlled the maturation of osteoclasts by regulating NFATc1 nuclear translocation. In contrast, by deleting Nron specifically in osteoclasts, tooth movement speed increased in Nron CKO mice. These results indicate that lncRNAs could be potential targets to regulate osteoclastogenesis and orthodontic tooth movement speed in the clinic in the future.
10.1038/s41368-020-0077-7
Transcriptional Expression in Human Periodontal Ligament Cells Subjected to Orthodontic Force: An RNA-Sequencing Study.
Kim Kyunam,Kang Hee Eun,Yook Jong In,Yu Hyung-Seog,Kim Euiseong,Cha Jung-Yul,Choi Yoon Jeong
Journal of clinical medicine
This study was performed to investigate the changes in gene expression in periodontal ligament (PDL) cells following mechanical stimulus through RNA sequencing. In this study, premolars extracted for orthodontic treatment were used. To stimulate the PDL cells, an orthodontic force of 100 was applied to the premolar (experimental group; = 11), whereas the tooth on the other side was left untreated (control group; = 11). After the PDL cells were isolated from the extracted teeth, gene set enrichment analysis (GSEA), differentially expressed gene (DEG) analysis, and real-time PCR were performed to compare the two groups. GSEA demonstrated that gene sets related to the cell cycle pathway were upregulated in PDL. Thirteen upregulated and twenty downregulated genes were found through DEG analysis. Real-time PCR results confirmed that five upregulated genes (CC2D1B, CPNE3, OPHN1, TANGO2, and UAP-1) and six downregulated genes (MYOM2, PPM1F, PCDP1, ATP2A1, GPR171, and RP1-34H18.1-1) were consistent with RNA sequencing results. We suggest that, from among these eleven genes, two upregulated genes, and , and one downregulated gene, , play an important role in PDL regeneration in humans when orthodontic force is applied.
10.3390/jcm9020358
Global gene expression profile of periodontal ligament cells submitted to mechanical loading: A systematic review.
Spitz Alice,Christovam Ilana Oliveira,Marañón-Vásquez Guido Artemio,Masterson Daniele Ferreira,Adesse Daniel,Maia Lucianne Cople,Bolognese Ana Maria
Archives of oral biology
OBJECTIVE:To evaluate the evidence reporting gene expression array data of human in vitro cultured periodontal ligament cells (PDLCs) submitted to static mechanical loading compared to a control group. DESIGN:Systematic searches were performed in MEDLINE/PubMed, Scopus, Web of Science, Virtual Health Library, The Cochrane Library and the System for Information on Grey Literature in Europe up to June 2019. A narrative synthesis was performed to summarize differentially expressed genes (DEGs). These were grouped according to the culture method (2D or 3D), force type (compression or tension) and observation time. Additionally, gene ontology (GO) analysis was performed using the Database for Annotation Visualization and Integrated Discovery. The risk of bias (RoB) and certainty of evidence (CoE) were assessed using a modified CONSORT checklist and the GRADE tool, respectively. RESULTS:Of eight studies included (all rated as having moderate RoB), only two provided the complete list of DEGs and four studies performed GO, gene network or pathways analysis. "Cell proliferation", "cell-cell signaling", "response to hypoxia and to mechanical stimulus" were among the significantly enriched biological processes in 3D-cultured compressed PDLCs (moderate CoE); while "collagen catabolic process", "extracellular matrix organization" and "cell proliferation" were associated with DEGs of 3D-cultured PDLCs submitted to tension (very low CoE). Biological processes significantly enriched in 2D-cultured PDLCs under compression were "extracellular matrix organization", "canonical glycolysis" and "glycolytic process" (very low CoE). CONCLUSION:Genes such as NR4A2, NR4A3, NAMPT, PGK1, and REDD1 are suggested as novel biomarkers for orthodontic tooth movement. Limited amount of evidence on the complete gene expression profile and the high heterogeneity in methodologies make it impossible to obtain definite conclusions. New studies following standardized and well-designed in vitro model and reporting complete gene expression datasets are needed.
10.1016/j.archoralbio.2020.104884
Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells.
Kanzaki Hiroyuki,Wada Satoshi,Yamaguchi Yuuki,Katsumata Yuta,Itohiya Kanako,Fukaya Sari,Miyamoto Yutaka,Narimiya Tsuyoshi,Noda Koji,Nakamura Yoshiki
BMC molecular and cell biology
BACKGROUND:Osteoclasts play a critical role in bone resorption due to orthodontic tooth movement (OTM). In OTM, a force is exerted on the tooth, creating compression of the periodontal ligament (PDL) on one side of the tooth, and tension on the other side. In response to these mechanical stresses, the balance of receptor activator of nuclear-factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) shifts to stimulate osteoclastogenesis. However, the mechanism of OPG expression in PDL cells under different mechanical stresses remains unclear. We hypothesized that compression and tension induce different microRNA (miRNA) expression profiles, which account for the difference in OPG expression in PDL cells. To study miRNA expression profiles resulting from OTM, compression force (2 g/cm) or tension force (15% elongation) was applied to immortalized human PDL (HPL) cells for 24 h, and miRNA extracted. The miRNA expression in each sample was analyzed using a human miRNA microarray, and the changes of miRNA expression were confirmed by real-time RT-PCR. In addition, miR-3198 mimic and inhibitor were transfected into HPL cells, and OPG expression and production assessed. RESULTS:We found that certain miRNAs were expressed differentially under compression and tension. For instance, we observed that miR-572, - 663, - 575, - 3679-5p, UL70-3p, and - 3198 were upregulated only by compression. Real-time RT-PCR confirmed that compression induced miR-3198 expression, but tension reduced it, in HPL cells. Consistent with previous reports, OPG expression was reduced by compression and induced by tension, though RANKL was induced by both compression and tension. OPG expression was upregulated by miR-3198 inhibitor, and was reduced by miR-3198 mimic, in HPL cells. We observed that miR-3198 inhibitor rescued the compression-mediated downregulation of OPG. On the other hand, miR-3198 mimic reduced OPG expression under tension. However, RANKL expression was not affected by miR-3198 inhibitor or mimic. CONCLUSIONS:We conclude that miR-3198 is upregulated by compression and is downregulated by tension, suggesting that miR-3198 downregulates OPG expression in response to mechanical stress.
10.1186/s12860-019-0187-2
Gene profiling of bone around orthodontic mini-implants by RNA-sequencing analysis.
Nahm Kyung-Yen,Heo Jung Sun,Lee Jae-Hyung,Lee Dong-Yeol,Chung Kyu-Rhim,Ahn Hyo-Won,Kim Seong-Hun
BioMed research international
This study aimed to evaluate the genes that were expressed in the healing bones around SLA-treated titanium orthodontic mini-implants in a beagle at early (1-week) and late (4-week) stages with RNA-sequencing (RNA-Seq). Samples from sites of surgical defects were used as controls. Total RNA was extracted from the tissue around the implants, and an RNA-Seq analysis was performed with Illumina TruSeq. In the 1-week group, genes in the gene ontology (GO) categories of cell growth and the extracellular matrix (ECM) were upregulated, while genes in the categories of the oxidation-reduction process, intermediate filaments, and structural molecule activity were downregulated. In the 4-week group, the genes upregulated included ECM binding, stem cell fate specification, and intramembranous ossification, while genes in the oxidation-reduction process category were downregulated. GO analysis revealed an upregulation of genes that were related to significant mechanisms, including those with roles in cell proliferation, the ECM, growth factors, and osteogenic-related pathways, which are associated with bone formation. From these results, implant-induced bone formation progressed considerably during the times examined in this study. The upregulation or downregulation of selected genes was confirmed with real-time reverse transcription polymerase chain reaction. The RNA-Seq strategy was useful for defining the biological responses to orthodontic mini-implants and identifying the specific genetic networks for targeted evaluations of successful peri-implant bone remodeling.
10.1155/2015/538080
Effect of corticision on orthodontic tooth movement in a rat model as assessed by RNA sequencing.
Gu Qihui,Guo Shuyu,Wang Dongyue,Zhou Tingting,Wang Lin,Wang Zhendong,Ma Junqing
Journal of molecular histology
Corticision is a common technique to accelerate orthodontic tooth movement; however, not much is known about the underlying mechanisms. In this study, we investigated the mechanism of alveolar tissue remodeling after corticision in a rat model of tooth movement (TM) by analyzing the differential transcriptome. A total of 36 male rats were equally divided into TM and TM with corticision (TM+C) groups. Alveolar bone response was examined using micro-computed tomography (micro-CT). Osteoclasts and osteoblasts were quantified on tartrate-resistant acid phosphatase (TRAP) and Goldner's trichrome staining. The transcriptomes of alveolus around the left maxillary first molar were determined on RNA sequencing (RNA-Seq), and the expression of selected differentially expressed genes (DEGs) validated on quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Immunohistochemical examination of alveolar tissue was performed to examine the expressions of correlative proteins of the selected signaling pathway in the TM and TM+C groups. The ratio of bone volume to total volume (BV/TV), and the trabecular number (Tb.N) were significantly decreased, while the movement distance and the trabecular separation (Tb.Sp) was significantly increased in the TM+C group. However, no significant between-group difference in trabecular thickness (Tb.Th) was observed. On histomorphometric analysis, a significant increase in the number of osteoclasts and increased bone resorption was observed in the TM+C group. A total of 399 DEGs were identified on RNA-SEq. Eleven selected genes were confirmed on qRT-PCR, which included components of the Ras signaling pathway. Four proteins of the Ras signaling pathway showed a higher expression in the TM+C group. Our findings indicate that corticision may speed up orthodontic tooth movement by accelerating osteoclastogenesis mediated via the Ras signaling pathway.
10.1007/s10735-017-9718-3
MicroRNA‑21 serves an important role during PAOO‑facilitated orthodontic tooth movement.
Zhang Yuanyuan,Tian Yulou,Yang Xiaofeng,Zhao Zhenjin,Feng Cuijuan,Zhang Yang
Molecular medicine reports
Periodontal accelerate osteogenesis orthodontics (PAOO) is an extension of described techniques that surgically alter the alveolar bone; however, the specific mechanism underlying the technique is not completely understood. The aim of the present study was to evaluate the roles of microRNA (miR)‑21 during PAOO. Sprague‑Dawley rats were divided into the following four groups: i) Group tooth movement (TM), underwent TM and were administered normal saline (NS); ii) Group PAOO, underwent PAOO + TM and were administered NS; iii) Group agomiR‑21, underwent PAOO + TM and were administered agomiR‑21; and iv) Group antagomiR‑21, underwent PAOO + TM and were administered antagomiR‑21. To validate the rat model of PAOO, morphological analyses were performed and measurements were collected. Reverse transcription‑quantitative PCR, western blotting and immunohistochemical staining were performed to examine the expression levels of programmed cell death 4 (PDCD4), activin A receptor type 2B (ACVR2b), receptor activator of NF‑κΒ ligand (RANKL) and C‑Fos. Dual‑luciferase reporter assays were performed to validate PDCD4 as a target of miR‑21 in vitro. Following 7 days of treatment, the TM distance of group PAOO was longer compared with groups TM and antagomiR‑21 (P<0.05), but shorter compared with group agomiR‑21 (P<0.05). Tartrate‑resistant acid phosphatase staining indicated that following treatment with agomiR‑21, osteoclast activity was notably increased, whereas the mRNA and protein expression levels of PDCD4 were notably decreased compared with group PAOO. The mRNA and protein expression levels of RANKL and C‑Fos in group agomiR‑21 were notably increased compared with group PAOO, whereas group antagomiR‑21 displayed the opposite pattern (P<0.05). With regard to ACVR2b, no significant differences were observed among the group agomiR‑21 and antagomiR‑21 compared with group PAOO. Bioinformatics analysis predicted that PDCD4 was a potential target gene of miR‑21, and dual‑luciferase reporter assays demonstrated that miR‑21 directly targeted PDCD4. In conclusion, the present study demonstrated that miR‑21 serves an important role during PAOO‑mediated orthodontic TM.
10.3892/mmr.2020.11107
Identification of Aberrantly Expressed lncRNAs Involved in Orthodontic Force Using a Subpathway Strategy.
Computational and mathematical methods in medicine
BACKGROUND:The aim of the study was to identify key long noncoding RNAs (lncRNA) and related subpathways in the periodontal ligament tissue following orthodontic force. METHODS:We adopt a novelty subpathway strategy to identify lncRNAs competitively regulated functions and the key competitive lncRNAs in periodontal ligament disorders after undergoing orthodontic force. To begin with, patients with orthodontics in our hospital were enrolled in our research. The relationship of lncRNA-mRNA was established through shared predicted miRNA by using the hypergeometric test, Jaccard coefficient standardization, and the Pearson coefficient to determine the valid interaction relationship. After embedding screened lncRNA interactions to pathways, the significant subpathways were recognized by lenient distance and Wallenius approximation methods to calculate the false discovery rate value of each subpathway. RESULTS:The lncRNA-mRNA intersections including 263 lncRNAs, 1,599 mRNAs, and 3,762 interacting pairs were obtained. The enriched mRNAs were further enriched into various candidate pathways such as the PI3K-Akt signaling pathway. Several subpathways were screened, including the PI3K-Akt signaling pathway, 04510_1 focal adhesion, and p53 signaling pathway, respectively. The network of pathway-lncRNA-mRNA was constructed. Several key lncRNAs including , , , and were screened. CONCLUSIONS:, , , and as aberrantly expressed lncRNAs involved in orthodontic force might play crucial roles in periodontal ligament disease pathogenesis.
10.1155/2019/9250129
Gene expression profile altered by orthodontic tooth movement during healing of surgical alveolar defect.
Choi Eun-Kyung,Lee Jae-Hyung,Baek Seung-Hak,Kim Su-Jung
American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics
INTRODUCTION:We explored the gene expression profile altered by orthodontic tooth movement (OTM) during the healing of surgical alveolar defects in beagles. METHODS:An OTM-related healing model was established where a maxillary second premolar was protracted into the critical-sized defect for 6 weeks (group DT6). As controls, natural healing models without OTM were set at 2 weeks (group D2) and at 6 weeks (group D6) after surgery. Total RNAs were extracted from dissected tissue blocks containing the regenerated defects and additionally from sound alveolar bone as a baseline (group C). mRNA profiling was performed using microarray analysis. RESULTS:Functional annotations of gene clusters based on differentially expressed genes among groups indicated that the gene expression profile of group DT6 had a stronger similarity to that of group D2 than to group D6. The genes participating in high woven-bone fraction in group DT6 could be identified as TNFSF11, MMP13, SPP1, and DMP1, which were verified by quantitative real-time polymerase chain reactions. CONCLUSIONS:We investigated at the gene level that OTM can affect the healing state of surgical defects serving as favorable matrices for OTM with defect regeneration. It would be a basis on selecting putative genes to be therapeutically applied for tissue-friendly accelerated orthodontics in the future.
10.1016/j.ajodo.2016.10.039