Supplementary Material for: microRNA-29a-3p, Up-Regulated in Human Gastric Cells and Tissues with H.Pylori Infection, Promotes the Migration of GES-1 Cells via A20-Mediated EMT Pathway

Background/Aims: Helicobacter pylori (H. pylori) infection is closely related to human gastric mucosa-associated diseases. Several recent studies on miRNAs have expanded our insights on H.pylori pathogenesis. This study aimed to investigate the biological roles and underlying molecular mechanisms of miR-29a-3p in human gastric cells and tissues with H.pylori infection. Methods: miR-29a-3p expression was quantified by quantitative RT-PCR (qRT-PCR). A miR-29a-3p target gene was validated by bioinformatics analysis, western blotting and dual luciferase reporter gene assays. Western blotting and immunohistochemistry (IHC) assay were performed to detect the protein expression. Transwell assay was used to determine the cell migration ability. Results: MiR-29a-3p was up-regulated in H.pylori-positive gastric mucosa tissues and H.pylori-infected gastric cells. The up-regulation of miR-29a-3p was dose-dependent in BGC-823 and GES-1 cells infected with H.pylori. Using gain- and loss-of-function experiments in vitro, we demonstrated that miR-29a-3p promoted the migration of gastric epithelial cells. We further characterized A20 as a direct target of miR-29a-3p. The expression of A20 was decreased in H.pylori-positive gastric mucosa tissues compared with H.pylori-negative gastric mucosa tissues. A20 downregulation was time- and dose-dependent in GES-1 and BGC-823 cells infected with H.pylori. In GES-1 and BGC-823 cells infected with H.pylori, the miR-29a-3p mimic significantly blocked A20 expression, which suggests that H.pylori decreased A20 expression through up-regulating miR-29a-3p in GES-1 and BGC-823 cells infected with H.pylori. The knockdown of A20 by siRNA enhanced the migration of human gastric epithelial cells and promoted the expression of Snail, Vimentin, and N-cadherin and inhibited the expression of E-cadherin. Conclusion: The miR-29a-3p may act as a tumor promotive miRNA by regulating cells migration through directly targeting of A20 gene in human gastric epithelial cells infected with H.pylori.