RNA-sequencing assay to Identify changed gene expression in esophageal squamous cell carcinoma (ESCC) cells with RAB23 knockdown

Purpose: To elucidate how RAB23 influenced the expression profile in ESCC cells, RAB23 expression was reduced using two siRNAs in ESCC KYSE30 cells. Then, transcriptional profiling of two populations of cells with RAB23 knockdown (si-RAB23-1 and si-RAB23-9) and the control cells (si-NC) was performed to characterize differentially expressed genes (DEG). Methods: When si-NC, si-RAB23-1 and si-RAB23-9 cells grew to approximately 80% confluence, these cells were washed twice using pre-chilled PBS. Total RNAs were harvested using Trizol reagents (Thermo Fisher Scientific). Poly(A) mRNA was isolated and enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module. The mRNA was then recovered for library generation with NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, E7420S) following the manufacturer's instructions. The cDNA libraries were sequenced at WuXiNextCODE (China). FastQC was used to examine the quality of raw reads. Read alignment was conducted using STAR (v2.5.1b), and R package edgeR (v3.8.5) was used to determine relative transcript abundances and differentially expressed genes (DEGs) between sample pairs. Results: Generally, 1041 genes were increased and 1013 genes decreased in si-RAB23-1 cells, compared with si-NC cells (FDR<0.05, Fold Change>2). Additionally, 1154 genes (FDR<0.05, Fold Change>2) were increased and 958 genes decreased in si-RAB23-9 cells, relative to si-NC cells. GO analysis showed that these changed genes were significantly enriched in signal pathways related to cancer. Conclusions: Reduced RAB23 expression alters multiple cancer-related signal pathways. Overall design: Two biological replicates for each group - si-NC, si-RAB23-1 and si-RAB23-9 - were subjected to RNA-sequencing analysis respectively, among which si-NC acted as the reference sample for si-RAB23-1 and si-RAB23-9.