Gene expression data from SQ-diEG-treated and BBN-induced bladder cancer mouse model at 8 and 16 W

Squalene(SQ), a natural triterpene with antioxidant, anti-inflammatory, and immunostimulatory properties, holds promise for cancer therapy. We examined a previously developed, diethylene glycol derivative of squalene (SQ-diEG) and investigated its in vivo anti-carcinogenic effects in bladder cancer. C57BL/6 mice(6–8 weeks old) were treated with 0.025% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) to induce bladder cancer, with SQ-diEG or PBS (control) administered orally from Week 0 to 8 or 16. On the last day of administration, mice was sacrificed and its bladder collect for the RNA extraction.We performed an untargeted whole-transcriptomic analysis to explore the functionality of SQ-diEG. Squalene(SQ), a natural triterpene with antioxidant, anti-inflammatory, and immunostimulatory properties, holds promise for cancer therapy. We examined a previously developed, diethylene glycol derivative of squalene (SQ-diEG) and investigated its in vivo anti-carcinogenic effects in bladder cancer. C57BL/6 mice(6–8 weeks old) were treated with 0.025% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) to induce bladder cancer, with SQ-diEG or PBS (control) administered orally from Week 0 to 8 or 16. On the last day of administration, mice was sacrificed and its bladder collect for the RNA extraction.We performed an untargeted whole-transcriptomic analysis to explore the functionality of SQ-diEG. According to the manufacturer's guide, the RNA was extracted using Isogen kit (Nippon Gene Co. Ltd., Japan). Then, RNA quantity and quality were determined using the NanoDrop 2000 spectrophotometer (ThermoScientific, USA). DNA microarray analysis was conducted on urinary bladder samples of control and SQ-diEG-treated groups using the GeneChip WT PLUS Reagent Kit (ThermoFisher Scientific) and GeneChip™ Hybridization, Wash and Stain Kit (ThermoFisher Scientific) following the manufacturer's instructions. In brief, complementary DNA (cDNA) was synthesized from 100 ng of RNA solutions. cRNA was synthesized from in vitro transcription of cDNA and then purified and reverse transcribed. Finally, single-stranded cDNA (ss-cDNA) was synthesized, purified, fragmented, and labeled following the manufacturer's instructions. Cartridge Array Hybridization was performed using the Clariom S array (Mouse; ThermoFisher Scientific) on the GeneChip™ Fluidics Station (ThermoFisher Scientific). Scanning was performed using GeneChip Scanner (ThermoFisher Scientific). The raw image data obtained after scanning were analyzed using the Transcriptome Analysis Console (TAC) software (ver. 4.0.2, ThermoFisher Scientific). The raw data were normalized following the signal space transformation robust multi-chip analysis (SST-RMA) algorithm. Further, gene-level analysis was performed using the Limma Bioconductor package. For differential expression analysis, a One-Way ANOVA followed by an empirical Bayes correction was performed. The detected above back-ground (DABG) cutoff was set to 0.05. The positive vs negative area under the curve (AUC) value was set at greater than or equal to 0.7. Finally, genes that passed the filter criteria of p value < 0.05 (one-way between-subjects ANOVA) and fold change > 2 (in linear space) were considered as differentially expressed genes (DEGs).