Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway.
OBJECTIVE
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.


CONCLUSIONS
Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.
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.
Xiao-Jun
XJ
Yang
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.
Ruo-Huang
RH
Si
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
YH
Liang
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.
Bing-Qiang
BQ
Ma
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.
Ze-Bin
ZB
Jiang
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.
Bin
B
Wang
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.
Peng
P
Gao
D020413:3' Untranslated Regions; D001343:Autophagy / Q000235:genetics; D000071192:Autophagy-Related Protein 12 / Q000235:genetics / Q000378:metabolism; D000071187:Autophagy-Related Protein 5 / Q000235:genetics / Q000378:metabolism; D000071183:Autophagy-Related Proteins / Q000235:genetics / Q000378:metabolism; D000071186:Beclin-1 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005753:Gastric Mucosa / Q000378:metabolism* / Q000382:microbiology / Q000648:ultrastructure; D015972:Gene Expression Regulation, Neoplastic; D016481:Helicobacter Infections / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D016480:Helicobacter pylori / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology / Q000648:ultrastructure; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; C466624:ATG12 protein, human; C572313:ATG2B protein, human; C502778:ATG5 protein, human; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071183:Autophagy-Related Proteins; C491997:BECN1 protein, human; C116838:BNIP3L protein, human; D000071186:Beclin-1; C546086:MIRN30b microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D033921:Vesicular Transport Proteins
Autophagy; Gastric cancer; Gene expression; Helicobacter pylori; mir-30d