RNA‐sequencing analysis reveals potential molecular mechanism of RAD54B in the proliferation of inflamed human dental pulp cells

alexandrossfakianakis shared this article with you from Inoreader RNA‐sequencing analysis reveals potential molecular mechanism of RAD54B in the proliferation of inflamed human dental pulp cells via International Endodontic Journal Abstract Aim To investigate the role of RAD54B in the proliferation of inflamed human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). Methodology Normal, carious, and pulpitic human dental pulp tissues were collected. Total RNA was subjected to RNA-sequencing (seq) and gene expression profiles were studied by Gene Ontology (GO) and KEGG pathway analysis. Differentially expressed genes (DEGs) in homologous recombination repair (HRR) were validated with qRT-PCR. The expression of RAD54B and TNF-α in human dental pulp tissues was detected using immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK-8 was used to investigate the proliferation of hDPCs transfected with negative control (Nc) small interfering RNA (siRNA), RAD54B siRNA, P53 siRNA or both siRNAs with or without LPS stimulation. Flow cytometry was used to detect the cell cycle distribution, and western blot and immunofluorescence were used to analyze the expression of RAD54B, P53 and P21 under the above treatments. One-way and two-way ANOVA followed by LSD posttest were used for statistical ana lysis. Results RNA-seq results identified DEGs among the three groups. KEGG pathway analysis revealed enrichment of DEGs in the replication and repair pathway. HRR and non-homologous end joining (NHEJ) components were further verified and qRT-PCR results were basically consistent with the sequencing data. RAD54B, an HRR accessory factor highly expressed in carious and pulpitic tissues as compared to that in the normal pulps, was chosen as our gene of interest. High RAD54B expression was confirmed in inflamed human dental pulp tissues and LPS-stimulated hDPCs. Upon RAD54B knockdown, P53 and P21 expressions in hDPCs were upregulated whereas the proliferation was significantly downregulated, accompanied by increased G2/M phase arrest. After inhibiting P53 expression in RAD54B-knockdown hDPCs, P21 expression and cell proliferation were reversed. Conclusions Gene expression profiles of normal, carious and pulpitic human dental pulp tissues were revealed. HRR components was elucidated to function in dental pulp inflammation. Among the DEGs in HRR, RAD54B regulated the proliferation of inflamed hDPCs via P53/P21 signalling. This research deepens understanding of dental pulp inflammation and provides a new insight to clarify the underlying mechanisms. View on Web

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