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RNA-sequencing assay to Identify changed gene expression in gastric cancer cells with SCAMP1 knockdown

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252526 | GEO | 2024/09/25

REPOSITORIES: GEO

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Project description:Background: Zinc finger and scan domain containing 18 (ZSCAN18) belongs to zinc finger transcription factor superfamily, which consists of the hundreds of members that play critical roles in all steps of tumorigenesis. Although hypermethylation of ZSCAN18 promoter and its consequent deficiency in a variety of malignancies has been reported recently, its functions and mechanisms in the initiation and progression of gastric cancer (GC) are not clear. This study aims to investigate the roles of ZSCAN18 in gastric carcinogenesis. Methods: Methylation of ZSCAN18 promoter in GC cell lines were analyzed via MassARRAY and treatment of 5-Aza-2'-deoxycytidine. Bioinformatics analysis, qRT-PCR, western blot, immunohistochemistry, CCK-8, colony formation, and in vivo xenograft tumor model were used to examine the expression and function of ZSCAN18. Survival analysis was performed to determine the correlation between ZSCAN18 expression and prognosis of patients with GC. Genes modulated by ZSCAN18 in NCI-N87 cells were identified through RNA next generation sequence screening and further verified by qRT-PCR in AGS and NCI-N87 cells. Results: ZSCAN18 expression was markedly reduced in GC tissues as compared with that in adjacent normal tissues due to hypermethylation in GC. Likewise, ZSCAN18 expression was also significantly reduced in a panel of GC cell lines as the result of densely methylated ZSCAN18 promoter. Forced expression of ZSCAN18 inhibited proliferation in AGS and NCI-N87 cells in vitro. Additionally, ZSCAN18 impaired growth of NCI-N87 cells in immunodeficient mice. Survival analysis showed that ZSCAN18 expression was positively associated with favorable outcome in patients with GC. RNA next generation sequence screening showed three representative genes (FGF20, CEACAM5 and TP53INP2) involved in downstream signaling pathways modulated by ZSCAN18. Conclusions: Collectively, this study unveils the anti-cancer role of ZSCAN18 in GC, providing a promising diagnostic and therapeutic target.

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RNA-sequencing assay to Identify changed gene expression in gastric cancer cells with SCAMP1 knockdown

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