Abstract No. 427 Osmotic stress blunted heat shock response in HepG2 cells

Abstract

Heat shock response may play a role in tumor progression after thermal ablations. Inhibition of these cytoprotective pathways may help to develop effective adjuvant therapy for thermal ablation. Our previous experiments showed that 200mM sodium acetate treatment decreased the protein stability of the master regulator of heat shock response, HSF1. We hypothesized that this treatment could inhibit HSF1 mediated transcriptional program in cells treated with concurrent thermal stress. To evaluate the effect of osmotic stress on hyperthermia-induced heat shock response, we designed this study to assess the transcriptome of HepG2 cells under combined osmotic and thermal stress by using RNA-seq. HepG2 cells were treated with 0.2M sodium acetate with or without concomitant hyperthermia (43°C, 2h). Total RNA was recovered by using a total RNA isolation kit (Qiagen), and mRNA was sequenced. About 20 million reads were obtained by using Illumina paired sequencing (PE150). Around 15,000 transcripts were identified for each sample. An RNA-seq pipeline at our institution was used to perform de novo assembly, genome alignment, and reads counting. HSP70 and HSP27 mRNA levels were also evaluated by qPCR to validate RNA-seq data. IPA analysis (Qiagen) was performed to identify signaling pathways affected by combined stress. About 6% of the transcriptome changed upon hyperthermia (± 2-fold). Many heat shock protein genes, including HSP70 and HSP27 were up-regulated. These changes were verified by qPCR. Protein folding/quality control and stress response pathways were activated, including protein ubiquitination pathway, unfolded protein response, and NRF2-mediated oxidative stress response. Osmotic stress up-regulated pathways involved in liver cell hyperplasia and proliferation. Adding osmotic stress to hyperthermia blunted expression of most heat shock protein genes that were otherwise induced by hyperthermia. Transcripts including non-coding RNA RN7SK, a non-specific transcription repressor, were increased in osmotic stress and heat shock, with the highest level seen in combined stress. Osmotic stress undermined the cytoprotective heat shock response by suppressing HSP expressions.