Abstract 784: Epigenetic silencing of miR490-3p by H. pylori activates DARPP-32 and induces resistance to gefitinib in gastric cancer cells

Abstract

Abstract Background: H. pylori causes epigenetic dysregulation to promote gastric carcinogenesis, but the roles and functions of microRNAs (miRNAs) in this multistage cascade are not fully explored. In this study, we proposed to investigate the expression and biological functions of miR490-3p in gastric cancer, as well as to identify its downstream target genes and pathways. Methods: miRNAs expression were profiled by RNAseq. Western blot analyses, Quantitative real-time PCR, ATP-Glo, clonogenic survival, Annexin V, and bisulfite PCR amplification were used. The survival rate was calculated using the Kaplan-Meier method and log-rank tests. Results: DARPP-32 was markedly upregulated in gastric cancer (P < 0.01), whereas, significant downregulation of miR-490-3p was confirmed in mice and human gastric cancer tissues (P < 0.05). Importantly, our clinical data demonstrated negative correlation of DARPP-32 and miR-490-3p in human gastric primary tumors, strongly supporting a role for miR-490-3p regulating DARPP-32 expression in gastric cancer (R = - 0.58, P < 0.01). miR490-3p host gene CHRM2 is hypermethylated and downregulated in mice and human gastric tissues (P < 0.05). Infection with H. pylori decreased the expression of miR490-3p in the in vitro and in vivo models (P < 0.05). qRT-PCR and Western blot analysis data showed higher protein and mRNA levels of DARPP-32 in H. Pylori infected mice than in control mice (P < 0.05). Reconstitution of miR490-3p in MKN45 cells sensitized cancer cells to gefitinib treatment by directly targeting DARPP-32, and inactivating AKT and STAT3 signaling pathways. Conversely, miR490-3p inhibitor increased DARPP-32 protein expression and reversed these signaling effects leading to decreased sensitivity to gefitinib, as measured by ATP-GLO Cell Viability and Clonogenic Survival Assay (P < 0.01). Knockdown of DARPP-32 significantly attenuated the pro-tumorigenic effects of miR490-3p inhibitor, whereas, enforced expression of DARPP-32 promoted gefitinib resistance in gastric cancer cells (P < 0.01). In survival analysis, gastric cancer patients with low levels of miR490-3p and high expression level of DARPP-32 had poor clinical outcome with decreased overall survival (P < 0.05). Conclusion: Hypermethylation-mediated silencing of CHRM2 and miR-490-3p by H. pylori increased DARPP-32 expression. Downregulation of miR-490-3p in gastric cancer plays a role in gefitinib response by inducing DARPP-32 and PI3K/AKT, STAT3 signaling pathways. Citation Format: Shoumin Zhu, Zheng Chen, Dunfa Peng, Tian-ling Hu, Heng Lu, Mohammed Soutto, Wael El-Rifai. Epigenetic silencing of miR490-3p by H. pylori activates DARPP-32 and induces resistance to gefitinib in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 784.