Figure 8 | Cell Death & Disease

Figure 8

From: Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients

Figure 8

Overactivation of the NF-κB pathway in inflammation is the main cause of dysregulation of the lncRNA-POIR and miR-182 regulatory network. (a) After transfection of anti-miR-182, lncRNA-POIR levels in hPDLSCs and pPDLSCs were measured by qPCR. (b) After transfection of shlncRNA-POIR, miR-182 levels in hPDLSCs and pPDLSCs were measured by qPCR. (c) Western blot was performed to detect the level of P65 in the cytoplasm and nucleus of hPDLSCs and pPDLSCs. β-Actin was used as the control for cytoplasmic P65 and HDAC1 was used as the control for P65 in the nucleus. (d) Schematic representation of the human pri-miR-182 promoter region in 2000 bp upstream of the transcription start site (TSS). ChIP assays were performed using input from cell lysate, normal mouse IgG, anti-P65 or anti-c-Rel. Relative expression levels of control and binding regions in pPDLSCs were detected by qPCR. Control: regions without binding sites of P65/c-Rel. Binding regions: regions with several binding sites of P65/c-Rel. (e) Transfection effect of siIKKα was determined by qPCR. (f and g) The pPDLSCs were transfected with IKKα SiRNA for 48 h and qPCR was performed. (h) Working model of lncRNA-POIR-miR-182 network in regulating osteogenesis of pPDLSCs. LncRNA-POIR and miR-182 could form a negative regulatory network and lead to a reduction of miR-182 target gene, FoxO1, which in turn inhibits canonical Wnt pathway. Besides, inflammation can increase miR-182 expression through the NF-κB pathway and the overexpressed miR-182 in the inflammatory microenvironment resulted in an imbalance in the lncRNA-POIR-miR-182 regulatory network. All experiments were repeated three times. Relative expressions of mRNAs and lncRNA-POIR were normalized by β-actin and relative expression of miR-182 was normalized by U6 in qPCR, respectively. Data represent mean±S.D. *P<0.05, **P<0.01 and NS, not significant. Anti-miR-NC, siPORT reagent alone; anti-miR-182, miR-182 inhibitor; Con, Control; shlncRNA-POIR, plamids for downregulating lncRNA-POIR; shNC, plasmids negative control; siNC, negative control; siIKKα, IKKα oligo

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