Dataset Information

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

ABSTRACT:

Background

Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).

Methods

Healthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-?-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R).

Results

HPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3'-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs.

Conclusion

This study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

SUBMITTER: Bian M

PROVIDER: S-EPMC7937634 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

Bian Minxia M Yu Yan Y Li Yuzhi Y Zhou Zhou Z Wu Xiao X Ye Xiaying X Yu Jinhua J

Frontiers in cell and developmental biology 20210222

<h4>Background</h4>Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).<h4>Methods</h4>Healthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolate ...[more]

PMID: 33692995

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Project description:The periodontium are the tissues supporting and investing the tooth and consists of the periodontal ligament, the gingiva, the root cementum, and the alveolar bone. The functions of the cell populations in health and disease regarding the host-mediated tissue destruction are not well understood. To get a first idea, of which genes might play a distinct role in chronic periodontal disease in vivo, we compared the genom-wide gene expressions of chronic inflamed and healthy periodontal ligament cells by microarray analysis and validated the data by real-time RT-PCR. The expression rates of 14.239 genes were investigated and 3.018 of them were found differentially expressed by at least two-fold, the expression rates of 1.451 genes were significantly up-regulated and the expression rates of 1.567 genes were significantly down-regulated in inflamed PDL cells. We focused on mainly structural components, for example, laminins and integrins, as well as degrading enzymes, for example, MMPs and cathepsins. The molecular composition of the laminin network varies in chronic inflamed compared to healthy PDL cells in vivo. Furthermore, integrin alpha6beta4, together with laminin-332, might be involved in chronic periodontal inflammation. Findings that diverse keratins were upregulated in chronic disease indicate that the epithelial cell rests of Malassez might also be involved in chronic periodontal inflammation. Also cathepsin B and cathepsin C might participate in the connective tissue destruction. The microarray analysis has identified a profile of genes potentially involved in chronic periodontal inflammation in vivo. Further studies are needed to entirely understand cellular activities during chronic periodontal inflammation in vivo. Experiment Overall Design: Periodontal tissue was collected from 32 patients at the Dental Medical School of the University of Goettingen (12 men, 20 women, aged between 18 and 72 years) from March 2005 to Dezember 2005. The tissue probes were taken from teeth, either extracted for orthodontic reasons (healthy periodontium) or because of chronic periodontal lesions. The differentiation between both collectives was performed using clinical and radiological parameters (clinical attachment loss, increase in probing depth, and radiographic bone loss). A detailed anamnesis of each patient was explored. All patients were without a medical history and were not on medication. In addition, the tissue samples used in our study were only taken from non-smokers. The extracted teeth were immediately frozen and stored at -80Â°C. All patients who participated in the study were informed about the nature and aim of this project and gave their written informed consent. The study was approved according to the regulations of the Ethics Committee of the Medical Faculty of the University of Goettingen.

Project description:BACKGROUND:Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. METHODS:Microarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DANCR was screened out. Its effects on proliferation and osteogenic differentiation was explored by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DANCR on cell growth curve. To quantify the effects of DANCR on osteogenic differentiation of PDLSCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed. RESULTS:Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of lncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers' upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. CONCLUSIONS:The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs' osteogenic differentiation.

Project description:The goal of this study was to compare the gene expression profiles of chronically inflamed human peri-implant and chronically inflamed human periodontal tissues in order to elucidate potential changes at the molecular level. Cells out of the pocket depth of the inflamed peri-implant and periodontal ligament as well as from the middle third of healthy periodontal ligament were applied. Genome-wide gene expression was compared with the help of microarray analysis, and the data were validated by real-time RT-PCR. The expression rates of 14,239 genes were investigated and 2,079 of them were found differentially expressed by at least two-fold; the expression rates of 1,093 genes were significantly up-regulated and the expression rates of 986 genes were significantly down-regulated in inflamed peri-implant cells compared to inflamed periodontal cells. We focused on genes coding for extracellular matrix components and those that degrade them. Only genes of non-fibril-forming collagens (types IV, VI, VII, and XVII) were increased in inflamed peri-implant tissue, whereas only the genes of two fibril-forming collagens (types III and XVII) were decreased, suggesting that peri-implant tissue re-models faster than periodontal tissue. Furthermore, cathepsin D and cathepsin S might participate to a greater extent in connective tissue destruction of peri-implant tissue. The present investigation demonstrated that, despite their clinical similarities, periimplantitis and periodontitis are two different diseases at the genetic level. Keywords: inflammation, peri-implant, periodontal ligament cells The peri-implant tissue was scraped of the implant and the periodontal tissue was scraped off the roots of the teeth. Both tissue probes were treated with the RNeasy mini Kit (Qiagen, Hilden, Germany) to isolate total RNA from chronically inflamed peri-implant and chronically inflamed PDL cells, according to the manufacturer’s instructions. Healthy PDL cells were taken from the middle third of the root clearly avoiding sampling the gingiva. The chronically inflamed peri-implant and periodontal cells were taken directly out of the depth of the pocket again avoiding the ingrown gingiva. Quality control and quantitation of total RNA samples were determined prior to the microarray experiments (Agilent 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA). Equal amounts of total RNA were pooled into an inflamed peri-implant and an inflamed and healthy periodontal cell pool. To monitor the relative abundance of mRNA for full-length human genes, the Affymetrix GeneChip HG_U133A was used, representing 22,283 probe sets (14,239 unique human genes). Labelled cRNA, fragmented to an average size of 100–150 bases, was hybridized to the GeneChips. For each pool of cRNA, two GeneChips were hybridized. Chronically inflamed peri-implant as well as healthy and chronically inflamed periodontal tissue was collected from 48 patients at the Dental Medical School of the University of Goettingen (21 men, 27 women, aged between 18 and 72 years) from March 2005 to August 2006, as already described by Gersdorff et al. (2007). The peri-implant tissue probes were taken from endosteal implants (Astra Tech®, ITI Bonefit®, or Brånemark®) with chronic peri-implant lesions (Schwarz et al. 2007). The periodontal tissue probes were taken from teeth, either extracted for orthodontic reasons or due to carious lesions (healthy periodontium) or from teeth with generalized chronic periodontitis (Armitage 2004). Teeth with healthy PDL had no clinical signs of inflammation (no bleeding on probing), exhibited a probing depth of ?3,5 mm and had no radiographic detectable bone loss. In contrast, implants and teeth with generalized chronic inflammation exhibited obvious clinical signals of inflammation (bleeding on probing), exhibited a probing depth of >4 mm and had radiographic evidence of bone loss (1 to 7 mm). A detailed anamnesis of each patient was explored. All patients were without a medical history, i.e. heart diseases or diabetes, and were not on medication. In addition, the tissue samples used in our study were only taken from non-smokers. The explanted implants and extracted teeth were immediately frozen and stored at -80 °C. All patients who participated in the study were informed about the nature and aim of this project and gave their written informed consent. The study was approved according to the regulations of the Ethics Committee of the Medical Faculty of the University of Goettingen.

Dataset Information

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

Background

Methods

Results

Conclusion

Publications

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

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