Abstract 201: Expression of miR-124 suppresses spermine oxidase-associated H2O2 generation in human gastric adenocarcinoma cells: Implications for infection/inflammation-induced carcinogenesis

Abstract

Abstract Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions such as Helicobacter pylori-associated gastritis and colitis-associated colon cancer. As a generator of H2O2, elevated SMOX contributes to the DNA damage and subsequent tumorigenesis associated with chronic inflammation. MicroRNA expression levels are also altered in inflammatory conditions. Specifically, the expression of miR-124, a potential negative regulator of SMOX, is silenced by DNA methylation in cases of ulcerative colitis and H. pylori-associated gastric cancer. We therefore sought to determine if the repressed levels of miR-124 expression that occur with inflammation are associated with the elevated levels of SMOX observed under these same conditions. In the current study, a miRNA mimic was used to overexpress miR-124-3p in AGS human gastric adenocarcinoma cells. Expression of SMOX mRNA and protein levels were then measured by qRT-PCR and Western blot analysis, and changes in SMOX activity were detected using a luminol-based assay to measure the production of H2O2. The resultant changes in intracellular polyamine levels were measured by HPLC. A bioinformatics analysis predicted the presence of a miR-124 recognition element in the 3′UTR of SMOX. To verify if miR-124 directly regulates SMOX mRNA, this portion of the SMOX 3′UTR was cloned downstream from the luciferase reporter gene, the resultant plasmid was cotransfected into AGS cells with the miR-124 mimic or a negative control, and luciferase activity was measured. Finally, the role of miR-124 expression in the regulation of SMOX induction resulting from H. pylori infection was investigated by infecting AGS cells with H. pylori in the presence or absence miR-124. The results of these experiments clearly indicate that miR-124 is a negative regulator of ROS-generating SMOX that is induced in chronic inflammation-associated tumorigenesis. In the AGS gastric cancer cell line, which harbors a highly methylated and silenced endogenous miR-124 gene, transfection with a miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 hours. Furthermore, this decreased SMOX activity in the presence of miR-124 resulted an increased level of intracellular spermine, the substrate of SMOX, which also serves as an important free-radical scavenger. It has become evident that elevated SMOX activity resulting from infectious agents and/or pro-inflammatory cytokines is likely a contributing factor in the development of inflammation-associated cancer; the results reported here indicate that this elevation is correlated with, and may result from, the loss of miR-124 expression that occurs during the progression from inflammation to cancer. Citation Format: Tracy Murray-Stewart, Johanna C. Sierra, Rupesh Chaturvedi, Keith T. Wilson, Robert A. Casero. Expression of miR-124 suppresses spermine oxidase-associated H2O2 generation in human gastric adenocarcinoma cells: Implications for infection/inflammation-induced carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 201. doi:10.1158/1538-7445.AM2015-201