Abstract
Overexpressed gastrin is reported to promote oncogenesis and development of gastric cancer by inhibiting apoptosis of cancer cells; however, the underlying mechanism remains unclear. Our study is aimed at revealing the mechanism underlying the effect of gastrin on apoptosis of gastric cancer cells. Gastrin-interfering cell line was constructed by stably transfecting gastrin-specific pshRNA plasmid to gastric cancer cell line BGC-823. Then, differentially expressed proteins between untreated BGC-823 and gastrin-interfering BGC-823 cell lines were detected by the iTRAQ technique. GO and KEGG analysis was used to analyze the differentially expressed genes that code these differentially expressed proteins. The Annexin V-FITC staining assay was used to detect gastric cancer cell apoptosis. The DCFH-DA fluorescent probe staining assay was used to measure intracellular ROS. Mitochondrial membrane potential was detected by flow cytometry. Western blot was used to analyze the mitochondria respiratory chain proteins and apoptosis-related proteins. A total of 107 differentially expressed proteins were identified by iTRAQ. GO and KEGG analysis showed that proteins coded by the corresponding differentially expressed genes were mainly enriched in the mitochondrial oxidative respiratory chain, and the expression of three proteins (COX17, COX5B, ATP5J) was upregulated. The three proteins with higher scores were verified by Western blot. The apoptosis rate of the gastrin knockdown cancer cell was significantly increased; meanwhile, gastrin knockdown leads to increase of membrane potential and decrease of intracellular ROS production. Additionally, Bax was significantly increased, whereas NF-κB-p65 and Bcl-2 were downregulated after knockdown of gastrin. Concomitantly, pretreatment with NAC reversed the effect of gastrin on the Bax and Bcl-2 expression. Gastrin promotes the production of ROS from mitochondria, activates NF-κB, and inhibits apoptosis via modulating the expression level of Bcl-2 and Bax.
Highlights
Gastrin is an important polypeptide hormone synthesized by gastroduodenal G cells and contributes to the regulation of a variety of gastrointestinal tract functions, including acid secretion, motility, and epithelial proliferation [1]
We found that the gastrin mRNA expression was significantly downregulated in two clones of pshRNAA1 and pshRNA-B3 (Figures 1(a) and 1(b))
Because the downregulation of the mRNA and protein expression level of gastrin was more significantly in pshRNA-A1 than that in pshRNA-B3, we selected the clone with stable pshRNA-A1 transfection to represent BGC823-gastrin KD for follow-up experiments
Summary
Gastrin is an important polypeptide hormone synthesized by gastroduodenal G cells and contributes to the regulation of a variety of gastrointestinal tract functions, including acid secretion, motility, and epithelial proliferation [1]. Despite the known role of gastrin as a normal gastrointestinal hormone, an increasing number of studies have shown its “dark side” [2,3,4,5,6,7,8,9,10]. Gastrin has been reported to be abnormally expressed in a variety of tumors, such as pancreatic cancer [3], colorectal cancer [4], lung cancer [5], and gastric cancer [6], and it can promote proliferation and metastasis of tumors [7], alter the immune cell signature in the tumor microenvironment [8], and inhibit cancer cell apoptosis [9, 10].
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