Abstract
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.
Highlights
Periodontitis is a chronic infectious disease of periodontal support tissue, during which osteoclast activates and leads to bone resorption (Hienz et al, 2015)
This study explores the role of Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL)/miR-7-5p/IGF1R axis in the osteogenic differentiation of inflamed periodontal ligament stem cells for the first time
The results indicated that inflamed periodontal ligament stem cells (iPDLSCs) had lower osteogenic activity compared with hPDLSCs, and lncRNA ANRIL might play a positive role in osteogenic differentiation potential
Summary
Periodontitis is a chronic infectious disease of periodontal support tissue, during which osteoclast activates and leads to bone resorption (Hienz et al, 2015). Increasing studies about periodontal ligament stem cells (PDLSCs) emerged as PDLSCs are considered to be the important seed cells of periodontal tissue regeneration and repair (An et al, 2016). PDLSCs are a type of tissue−specific mesenchymal stem cells (MSC) and have corresponding specialties, such as self−renewal, multipotency, immunosuppressive response. They can differentiate into chondrogenic, adipogenic, and osteogenic lineages in vivo and in vitro (Wada et al, 2009; Tsumanuma et al, 2011; Nunez et al, 2019). This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs)
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