IADR Abstract Archives
MicroRNA Global-Profiling in Pulp Cells Identifies Candidates for Epigenetically-Driven Tissue-Repair
Objectives: MicroRNAs (miRNAs) regulate cellular-processes including epigenetic modifications.
Aim: To investigate global miRNA-expression during DPC-differentiation, and to examine the additional effect of two epigenetic-modifiers, the histone-deacetylase-inhibitor (HDACi; SAHA), and a DNA-methyl-transferase-inhibitor (DNMTi; 5-AZA-CdR), on DPC-proliferation, mineralisation and miRNA-expression.
Methods: SAHA (1µM) and 5-AZA-CdR (1µM) were applied to rat-DPCs and reparative-processes were investigated using proliferation (cell-count, MTT) and mineralisation (Alizarin-Red) assays. MiRNA expression was assessed by RNA-seq analysis at 4-days and validated by qRT-PCR (rat/human-DPCs). Ontological-profiling (StrandNGS), target-gene-prediction (TargetScan/miRDB/DIANA tools) and pathway-analysis (DIANA-tools, DAVID-Bioinformatics) were undertaken after removing redundant-data to identify novel miRNAs and pathways.
Results: SAHA and 5-AZA-CdR increased mineralisation at 11, 14-days, but reduced DPC-proliferation compared with induced-controls. Bioinformatic-RNA-seq-analysis identified 487 known and 11,969 novel-miRNAs. Differential-expression analysis (>1.5-fold-change[FC]) identified 46 miRNAs in mineralising-DPCs compared with non-mineralising controls. Comparing mineralising SAHA-treated cultures with controls identified 103 significantly differentially-regulated miRNAs. 5-AZA-CdR-treated samples showed 24 differentially regulated miRNAs. Selected miRNAs were further analysed if dysregulated in all three mineralising-groups or both epigenetic-modifier groups, had a previous link to epigenetic or mineralisation-processes and had human-orthologs. Four dysregulated miRNA-200b-3p, miR-205, miR-182 and miR-221-5p were validated by qRT-PCR in rat and human DPCs. Target gene prediction of miR-205, miR-182 identified genes linked to reparative-processes, including SMAD1 and SMAD4. Pathway-analysis identified several significantly dysregulated-pathways including TGF-β and Wnt-signalling.
Conclusions: Promotion of mineralisation in HDACi and DNMTi DPC-cultures was associated with an increase in several differentially-expressed miRNAs. MiRNA were linked to mineralisation-associated genes/pathways and validated in human cultures, highlighting the potential application for miRNA epigenetic-based therapeutics in regenerative endodontics.
Aim: To investigate global miRNA-expression during DPC-differentiation, and to examine the additional effect of two epigenetic-modifiers, the histone-deacetylase-inhibitor (HDACi; SAHA), and a DNA-methyl-transferase-inhibitor (DNMTi; 5-AZA-CdR), on DPC-proliferation, mineralisation and miRNA-expression.
Methods: SAHA (1µM) and 5-AZA-CdR (1µM) were applied to rat-DPCs and reparative-processes were investigated using proliferation (cell-count, MTT) and mineralisation (Alizarin-Red) assays. MiRNA expression was assessed by RNA-seq analysis at 4-days and validated by qRT-PCR (rat/human-DPCs). Ontological-profiling (StrandNGS), target-gene-prediction (TargetScan/miRDB/DIANA tools) and pathway-analysis (DIANA-tools, DAVID-Bioinformatics) were undertaken after removing redundant-data to identify novel miRNAs and pathways.
Results: SAHA and 5-AZA-CdR increased mineralisation at 11, 14-days, but reduced DPC-proliferation compared with induced-controls. Bioinformatic-RNA-seq-analysis identified 487 known and 11,969 novel-miRNAs. Differential-expression analysis (>1.5-fold-change[FC]) identified 46 miRNAs in mineralising-DPCs compared with non-mineralising controls. Comparing mineralising SAHA-treated cultures with controls identified 103 significantly differentially-regulated miRNAs. 5-AZA-CdR-treated samples showed 24 differentially regulated miRNAs. Selected miRNAs were further analysed if dysregulated in all three mineralising-groups or both epigenetic-modifier groups, had a previous link to epigenetic or mineralisation-processes and had human-orthologs. Four dysregulated miRNA-200b-3p, miR-205, miR-182 and miR-221-5p were validated by qRT-PCR in rat and human DPCs. Target gene prediction of miR-205, miR-182 identified genes linked to reparative-processes, including SMAD1 and SMAD4. Pathway-analysis identified several significantly dysregulated-pathways including TGF-β and Wnt-signalling.
Conclusions: Promotion of mineralisation in HDACi and DNMTi DPC-cultures was associated with an increase in several differentially-expressed miRNAs. MiRNA were linked to mineralisation-associated genes/pathways and validated in human cultures, highlighting the potential application for miRNA epigenetic-based therapeutics in regenerative endodontics.