scholarly journals miRNA-125b Regulates Osteogenic Differentiation of Periodontal Ligament Cells Through NKIRAS2/NF-κB Pathway

2018 ◽  
Vol 48 (4) ◽  
pp. 1771-1781 ◽  
Author(s):  
Nan Xue ◽  
Lin Qi ◽  
Guorong Zhang ◽  
Yang Zhang
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Osteoblast Differentiation ◽  
Bone Repair ◽  
Alveolar Bone ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Glycerol Phosphate

Background/Aims: Osteogenesis of periodontal ligament cells (PDLCS) is essential for alveolar bone repair. Varieties of factors have been found involved in the regulation of PDLCs osteoblast differentiation. Aim of this study was to identify microRNA as a regulator of the os-teogenic differentiation of PDLCs. Methods: The CD markers were analyzed by flow cytometry analysis. Osteoblast differentiation of PDLCs was induced by treatment with dexamethasone, β-glycerol phosphate and α-ascorbic acid. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by RT-qPCR and Western Blot, and mineralization at the same time. MicroRNA and NKIRAS2 expression was determined by RT-qPCR. Luciferase reporter assays were performed to test whether miR-125b is capable of interacting with the 3’UTR sequence of NKIRAS2. The possible signaling pathway was determined by Western Blot. Results: In this study, we found that the expression of miR-125b was down regulated during the process of ostoblast differentiation of PDLCs. When the expression of miR-125b was up regulated, the osteogenic differentiation of PDLCs was inhibited. During this process, the over-expressed miR-125b led to the activation of NF-κB. NF-κB inhibitor interacting RAS-like 2 (NKIRAS2) is one of target gene of miR-125b, and it is a regulator of NF-κB signaling that plays various roles in osteoblastic differentiation. We demonstrate thatmiR-125b is involved in osteogenic differentiation of PDLCs. Conclusion: Our data support the hypothesis that that miR-125b attenuates PDLCs osteoblastic differentiation by targeting NKIRAS2 and enhancing NF-κB signaling.

scholarly journals Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Xinwei Liu ◽  
Yue Zhou
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Reporter Assay ◽  
Protein Levels ◽  
Alp Activity ◽  
Dual Luciferase Reporter Assay

Background. Long noncoding RNAs (lncRNAs) are dysregulated in periodontitis development and involved in osteogenesis. The current study was aimed at investigating the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms. Methods. Firstly, the level of ANRIL was tested by qPCR. Then, PDLCs were treated with a mineralizing solution to induce osteogenic differentiation. ALP activity was measured, and protein levels of BMP2, Osterix, and OCN were measured by Western blot. A target of ANRIL was verified using dual-luciferase reporter assay. miR-7 level was measured by qPCR, and the signals of the NF-κB pathway were tested by Western blot. Results. ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were increased to show that PDLCs were differentiated. ANRIL level was increased in differential PDLCs, in which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7. Conclusions. Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

scholarly journals Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway

2021 ◽  
Author(s):  
Xinwei Liu ◽  
Yue Zhou
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Protein Levels ◽  
Alp Activity ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Abstract Background Long non-coding RNAs (lncRNAs) are dysregulation in periodontitis development and involved in osteogenesis. The current study aimed was to investigate the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms.Methods Firstly, the level of ANRIL was tested by qPCR. Then PDLCs were treated with a mineralizing solution to induce the osteogenic differentiation. ALP activity was measured and protein levels of BMP2, Osterix, and OCN were measured by western blot. A target of ANRIL was verified using dual-luciferase reporter assay. MiR-7 level was measured by qPCR and the signalings of NF-κB pathway were tested by western blot.Results ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were reduced to show PDLCs were differentiated. ANRIL level was increased in differential PDLCs, and which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7.Conclusions Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

scholarly journals CircRNA FAT1 Regulates Osteoblastic Differentiation of Periodontal Ligament Stem Cells via miR-4781-3p/SMAD5 Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Yu Ye ◽  
Yue Ke ◽  
Liu Liu ◽  
Tong Xiao ◽  
Jinhua Yu
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Periodontal Regeneration ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Mirna Sponge ◽  
Proliferation Capacity

The ability of human periodontal ligament stem cells (PDLSCs) to differentiate into osteoblasts is significant in periodontal regeneration tissue engineering. In this study, we explored the role and mechanism of circRNA FAT1 (circFAT1) in the osteogenic differentiation of human PDLSCs. The proliferation capacity of PDLSCs was evaluated by EdU and CCK-8 assay. The abilities of circFAT1 and miR-4781-3p in regulating PDLSC differentiation were analyzed by western blot, reverse transcription-polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP), and Alizarin red staining (ARS). A nucleocytoplasmic separation experiment was utilized for circFAT1 localization. A dual-luciferase reporter assay confirmed the binding relationship between miR-4781-3p and circFAT1. It was showed that circFAT1 does not affect the proliferation of PDLSCs. The osteogenic differentiation of PDLSCs was benefited from circFAT1, which serves as a miRNA sponge for miR-4781-3p targeting SMAD5. Both knockdown of circFAT1 and overexpression of miR-4781-3p suppressed the osteogenic differentiation of PDLSCs. Thus, circFAT1 might be considered as a potential target of PDLSCs mediated periodontal bone regeneration.

scholarly journals Cyclic Stretch Enhances Osteogenic Differentiation of Human Periodontal Ligament Cells via YAP Activation

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Yang Yang ◽  
Bei-Ke Wang ◽  
Mao-Lin Chang ◽  
Zi-Qiu Wan ◽  
Guang-Li Han
Keyword(s):  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Alveolar Bone ◽  
Tooth Movement ◽  
Nuclear Translocation ◽  
Cyclic Stress ◽  
Cyclic Stretch ◽  
Periodontal Ligament Cells ◽  
Human Periodontal Ligament Cells ◽  
Orthodontic Forces

Periodontal remodeling and alveolar bone resorption and formation play essential roles during orthodontic tooth movement (OTM). In the process, human periodontal ligament cells (HPDLCs) sense and respond to orthodontic forces, contributing to the alveolar bone formation. However, the underlying mechanism in this process is not fully elucidated. In the present study, cyclic stress stimulus was applied on HPDLCs to mimic the orthodontic forces during OTM. Our results demonstrated that cyclic stretch promoted the osteogenic differentiation of HPDLCs. Moreover, our data suggested that yes-associated protein (YAP), the Hippo pathway effector, which also involved in mechanical signaling transduction, was activated as we found that the nuclear translocation of YAP was significantly increased in the cyclic stress treated HPDLCs. The mRNA expression of CTGF and CYR61, the target genes of YAP, was also remarkably increased. Furthermore, knockdown of YAP suppressed the cyclic stretch induced osteogenesis in HPDLCs, while overexpression of YAP in HPDLCs enhanced osteogenesis. We also noticed that YAP activities could be suppressed by the ROCK and nonmuscle myosin II inhibitors, Y-27632 and Blebbistatin. The inhibitors also significantly inhibited the cyclic stretch induced osteogenesis in HPDLCs. Finally, in the murine OTM model, our results revealed that YAP was upregulated and nuclearly translocated in the PDLCs at the tension side. In summary, our present study demonstrated that cytoskeleton remodeling induced activation of YAP signaling pathway was crucial for the cyclic stretch-induced osteogenesis of HPDLCs, which might play important roles during OTM.

scholarly journals Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Manman Ren ◽  
Ya Zhao ◽  
Zhiqi He ◽  
Jian Lin ◽  
Chuchu Xu ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Mapk Signaling ◽  
Periodontal Ligament Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alizarin Red ◽  
Osteogenic Markers ◽  
Anti Inflammatory

Abstract Background Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS). Methods Human PDLCs were incubated with baicalein (0–100 μM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot. Results MTT results showed that baicalein up to 100 μM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/β-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs. Conclusions With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

Recombinant Human Bone Morphogenetic Protein-2 Stimulates Osteoblastic Differentiation in Cells Isolated from Human Periodontal Ligament

1999 ◽  
Vol 78 (10) ◽  
pp. 1624-1633 ◽  
Author(s):  
M. Kobayashi ◽  
T. Takiguchi ◽  
R. Suzuki ◽  
A. Yamaguchi ◽  
K. Deguchi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Periodontal Ligament ◽  
Osteoblast Differentiation ◽  
Periodontal Regeneration ◽  
Osteoblastic Differentiation ◽  
Bone Morphogenetic Protein 2 ◽  
Periodontal Ligament Cells ◽  
Morphogenetic Protein ◽  
Human Bone Morphogenetic Protein ◽  
Recombinant Human Bone

Periodontal ligament cells may play an important role in the successful regeneration of the periodontium. We investigated the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2), one of the most potent growth factors that stimulates osteoblast differentiation and bone formation, on cell growth and osteoblastic differentiation in human periodontal ligament cells (HPLC) isolated from four adult patients. rhBMP-2 induced no significant changes in cell growth in any of the HPLCs. rhBMP-2 at concentrations over 50 ng/mL significantly stimulated alkaline phosphatase (ALPase) activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in the HPLCs. rhBMP-2 (500 ng/mL) also slightly enhanced the level of PTH/PTH-related peptide receptor mRNA expression in these cells. While interleukin-1β enhanced ALPase activity stimulated with rhBMP-2, tumor necrosis factor-a inhibited the rhBMP-2-stimulated activity. Interleukin-6 induced no significant changes in ALPase activity stimulated with rhBMP-2. Although HPLCs, whether treated with rhBMP-2 or not, could not produce measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced osteocalcin mRNA expression and protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HPLCs at both the mRNA and protein levels. These results suggest that rhBMP-2 provides an anabolic effect on periodontal regeneration by stimulation of osteoblastic differentiation in human periodontal ligament cells, and that this stimulatory effect is differentially modulated by inflammatory cytokines during the course of periodontal regeneration.

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

2021 ◽  
Vol 9 ◽  
Author(s):  
Minxia Bian ◽  
Yan Yu ◽  
Yuzhi Li ◽  
Zhou Zhou ◽  
Xiao Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Non Coding Rna ◽  
Protein Expressions

BackgroundLong 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).MethodsHealthy 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)-β-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).ResultsHPDLSCs 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.ConclusionThis study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells

2014 ◽  
Vol 88 (2-3) ◽  
pp. 33-41 ◽  
Author(s):  
Tadashi Yamamoto ◽  
Yuki Ugawa ◽  
Keisuke Yamashiro ◽  
Masayuki Shimoe ◽  
Kazuya Tomikawa ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Rho Kinase ◽  
Periodontal Ligament Cells
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