RNA-sequencing assay to Identify changed gene expression in stomach adenocarcinoma (STAD) cells with SMYD3 knockdown

Purpose: To elucidate how SMYD3 influenced the expression profile in STAD cells, SMYD3 expression was reduced using one shRNA in STAD HGC-27 and SGC-7901 cells. Then, transcriptional profiling of cells with SMYD3 knockdown (shSMYD3) and the control cells (shNC) was performed to characterize differentially expressed genes (DEG).Methods: When shNC and shSMYD3 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, 248 genes were increased and 112 genes decreased in HGC-27 shSMYD3 cells, compared with shNC cells (FDR<0.05, Fold Change>2). Additionally, 135 genes (FDR<0.05, Fold Change>2) were increased and 97 genes decreased in SGC-7901 shSMYD3 cells, relative to shNC cells. GO analysis showed that these changed genes were significantly enriched in signal pathways related to cancer. Conclusions: Reduced SMYD3 expression alters multiple cancer-related signal pathways.