Huang Pei, Wang Fushi, Wang Xinhuan, Meng Xiujiao, Qiao Weiwei, Meng Liuyan
The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
Int Endod J. 2023 Jan;56(1):39-52. doi: 10.1111/iej.13842. Epub 2022 Oct 13.
To investigate the role of RAD54B in the proliferation of inflamed human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS).
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 Kyoto Encyclopedia of Genes and Genomes (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 analyse the expression of RAD54B, P53 and P21 under the above treatments. One-way and two-way anova followed by least significant difference posttest were used for statistical analysis.
RNA-seq results identified DEGs amongst 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 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.
Gene expression profiles of normal, carious and pulpitic human dental pulp tissues were revealed. HRR components were elucidated to function in dental pulp inflammation. Amongst the DEGs in HRR, RAD54B regulated the proliferation of inflamed hDPCs via P53/P21 signalling. This research deepens our understanding of dental pulp inflammation and provides new insight to clarify the underlying mechanisms.
研究RAD54B在脂多糖（LPS）诱导的人牙髓细胞（hDPCs）增殖中的作用。
收集正常、龋损和牙髓炎患者的人牙髓组织。提取总RNA进行RNA测序（seq），并通过基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路分析研究基因表达谱。采用qRT-PCR验证同源重组修复（HRR）中的差异表达基因（DEGs）。用免疫组织化学法检测人牙髓组织中RAD54B和TNF-α的表达。培养hDPCs，用蛋白质免疫印迹法检测LPS刺激后hDPCs中RAD54B的水平。用CCK-8法研究转染阴性对照（Nc）小干扰RNA（siRNA）、RAD54B siRNA、P53 siRNA或两种siRNA的hDPCs在有无LPS刺激下的增殖情况。用流式细胞术检测细胞周期分布，用蛋白质免疫印迹法和免疫荧光法分析上述处理下RAD54B、P53和P21的表达。采用单因素和双因素方差分析及最小显著差异事后检验进行统计学分析。
RNA测序结果在三组中鉴定出DEGs。KEGG通路分析显示DEGs在复制和修复通路中富集。进一步验证了HRR和非同源末端连接（NHEJ）成分，qRT-PCR结果与测序数据基本一致。RAD54B是一种HRR辅助因子，与正常牙髓相比，在龋损和牙髓炎组织中高表达，被选为我们感兴趣的基因。在炎症牙髓组织和LPS刺激的hDPCs中证实了RAD54B的高表达。敲低RAD54B后，hDPCs中P53和P21的表达上调，但增殖显著下调，同时G2/M期阻滞增加。在敲低RAD54B的hDPCs中抑制P53表达后，P21表达和细胞增殖得到逆转。
揭示了正常、龋损和牙髓炎患者人牙髓组织的基因表达谱。阐明了HRR成分在牙髓炎症中的作用。在HRR的DEGs中，RAD54B通过P53/P21信号通路调节炎症hDPCs的增殖。本研究加深了我们对牙髓炎症的理解，并为阐明其潜在机制提供了新的见解。
