OR12 - Autophagy Signaling Pathways in Dental Pulp Stem Cells Under Hypoxic Condition Using Rna-seq

Abstract: Introduction: Success of vital pulp therapy depends on Dental Pulp Stem Cell (DPSC) survival, differentiation into odontoblast and deposition of matrix for mineralization of dentin. Autophagy is a cellular catabolic pathway involved in protein degradation, organelle turnover, and non-selective breakdown of cytoplasmic components. The objective of this research is to investigate key gene candidates involved in cellular catabolic pathways associated with macroautophagy in human dental pulp tissue and DPSC. Methods: DPSCs isolated from human dental pulp were cultured in a hypoxic chamber (3%) for 21 days at 37°C in differentiation media and controls were cultured under normal conditions. Later, DPSCs collected were extracted for mRNA isolation. The mRNA was sequenced using NextSeq Illunima and was analyzed using the ERGO transcription tool with statistical methods such as Limma and DESeq2. Results: Total of 35 biomarkers were identified that are up regulated (6-fold) under hypoxic conditions compared to normal conditions. These include genes for CTSD, CTSB, LAP1, SQSTM1, IRS2, GABARAP, RAB7A. The genes and pathways were identified in KEGG pathways overlaying expression values. Conclusions: Autophagy is regulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation and ER stress. A robust quantification of gene expression using RNA-Seq technology to investigate key gene candidates involved with macroautophagy metabolic pathways was developed. In combination with power statistical analyses such as DESeq2 and Limma, certain metabolic pathways may be identified that are responsible for starvation, growth factor deprivation and ER stress as key diagnostic markers for pulp tissue diagnostics.