Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 21, 2016; 22(15): 3978-3991
Published online Apr 21, 2016. doi: 10.3748/wjg.v22.i15.3978
Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway
Xiao-Jun Yang, Ruo-Huang Si, Yu-He Liang, Bing-Qiang Ma, Ze-Bin Jiang, Bin Wang, Peng Gao
Xiao-Jun Yang, Ruo-Huang Si, Bing-Qiang Ma, Ze-Bin Jiang, Bin Wang, Peng Gao, Department of General Surgery, Gansu Provincial Hospital, Lanzhou 730000, Gansu Province, China
Yu-He Liang, Department of General Surgery, The People’s Hospital of Baoji City, Baoji 721000, Shaanxi Province, China
Author contributions: Yang XJ, Si RH and Liang YH contributed equally to this work; Yang XJ and Liang YH carried out cell culture, transfection and the molecular genetic studies, drafted the manuscript; Si RH carried out the RT-PCR; Ma BQ and Jiang ZB participated in the GFP-LC3 Plasmid Transfection and Flow cytometry; Wang B performed the statistical analysis; and Gao P conceived of the study, and participated in its design and coordination and helped to draft the manuscript.
Supported by the National Natural Science Fund from China, No. 81260326.
Institutional review board statement: This study was reviewed and approved by Gansu Provincial Hospital Institutional review board.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional animal care and use committee of Gansu Provincial Hospital.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Peng Gao, MD, Professor, Chief, Department of General Surgery, Gansu Provincial Hospital, No. 204 Donggang West Road, Lanzhou 730000, Gansu Province, China.
Telephone: +86-931-8281015 Fax: +86-931-8266957
Received: December 7, 2015
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.

Keywords: mir-30d, Helicobacter pylori, Autophagy, Gene expression, Gastric cancer

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.