Published online Apr 21, 2016. doi: 10.3748/wjg.v22.i15.3978
Peer-review started: December 10, 2015
First decision: December 30, 2015
Revised: January 14, 2016
Accepted: February 20, 2016
Article in press: February 22, 2016
Published online: April 21, 2016
AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival.
METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay.
RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3′ untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P < 0.05, vs control cells without mir-30d mimic treatment). Mir-30d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells.
CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.
Core tip: In this study, we tested a hypothesis that mir-30d could repress autophagy in response to Helicobacter pylori (H. pylori) invasion by directly targeting multiple core genes of the autophagy pathway, including ATG2B, ATG5, ATG12, BECN1 and BNIP3L in gastric epithelial cells. Inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells, and the repression of autophagy by mir-30d may help the intracellular H. pylori to evade autophagic clearance. These findings provide a novel mechanism for elucidating persistent H. pylori infection and provide a promising target for gastric cancer prevention.