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RNA-seq of YAP's role in hDPCs.

ABSTRACT: We report the application of RNA sequencing technology for high-throughput profiling of gene expression in YAP2SA-lentivirus-infected adult human dental pulp stromal cells (hDPCs).To overexpress YAP2SA in hDPCs we transfected cells with YAP2SA-lentivirus. Cells were derived from a 18~22-year-old healthy male human donor. Total mRNA was collected using TRIzol ® (Invitrogen, CA, USA) solution. Then all the extraction procedures were conducted according to manufacture’s instructions for RNA extraction from homogenized tissues. After quality checks they were subjected to the high-throughput RNA-sequencing. Every group consists of 3 replicates which were from 3 independent experimental repeats.

ORGANISM(S): Homo sapiens

PROVIDER: GSE213339 | GEO | 2023/09/20

REPOSITORIES: GEO

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Project description:Purpose: To compare the transcriptional changes of genes in dental pulp tissues with different degrees of inflammatory severity and investigate the role of RAD54B in inflamed human dental pulp cells (hDPCs) Methods: Normal, carious, and pulpitis human dental pulp tissues were collected. Total RNA extracted were subjected to RNA-sequencing and gene expression profiles were further studied by Gene Ontology (GO) and KEGG pathway analysis. DEGs (differentially expressed genes) in homologous recombination repair (HRR) were validated with qRT-PCR. The expression of RAD54B and TNF-α in human dental pulp tissues was detected by immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK-8 was applied to investigate the cell 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 applied to detect the cell cycle distribution, and western blot and immunofluorescence were utilized 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 analysis. Results: RNA-sequencing results identified DEGs among three groups. KEGG pathway analysis revealed enrichment of DEGs in 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, a HRR accessory factor highly expressed in carious and pulpitis tissues compared to normal pulp, 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 cell proliferation was significantly downregulated, accompanied with 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 pulpitis human dental pulp tissues were revealed. HRR components was elucidated to function in dental pulp inflammation. Among HRR DEGs, RAD54B could regulate the cell proliferation of inflamed hDPCs via P53/P21 signaling. This research not only deepens our understanding of dental pulp inflammation but also provides a new insight to clarify the underlying mechanisms.

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RNA-seq of YAP's role in hDPCs.

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