RNA-sequencing assay to Identify changed gene expression in gastric cancer cells with SCAMP1 knockdown
Purpose: To understand how SCAMP1 drives the proliferation of GC cells,  endogenous SCAMP1 expression was reduced using shRNA in gastric cancer cell line NCI-N87. RNA sequencing was performed to characterize differentially expressed genes (DEGs) betwenn the control (shCtrl) and the SCAMP1-depleted (shS1#1) NCI-N87 cells. Methods: When shCtrl and shS1#1 NCI-N87 cells grew to approximately 80% confluence, these cells were washed twice using pre-chilled PBS. Total RNAs were extracted using Trizol reagents (Thermo Fisher Scientific).  Poly(A) mRNAs were isolated and enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library was generated with NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, E7420S) following the manufacturer's instruction, and library was then sequenced by Novogene (Beijing, 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 abundance and differentially expressed genes (DEGs) between shCtrl and shS1#1 sample. Results: Generally, the expressions of 495 genes increased whereas the expressions of 204 genes decreased in shS1#1 cells, compared with shCtrl cells (|log2FC| > 1 and P < 0.05). Gene Ontology (GO) analysis indicated that significant portion of these differentially expressed genes were enriched in ligand-receptor interactions, such as fibroblast growth factor receptor binding, and the downstream signaling pathways, such as PI3K-Akt pathway. Conclusions: Reduced SCAMP1 expression attenuates the proliferation in GC cells via deactivating multiple pro-tumoral signaling pathways. Overall design: Three biological replicates for each group - shCtrl and shS1#1 - were subjected to RNA-sequencing analysis respectively, among which  shCtrl acted as the reference sample for shS1#1 .