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

Abstract

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: Autophagy; Gastric cancer; Gene expression; Helicobacter pylori; mir-30d.

Figure 1

Autophagy and mir-30d are upregulated in AGS and GES-1 cell lines in response to Helicobacter pylori infection. A: GFP-LC3 puncta were observed in AGS cells with or without 24 h Helicobacter pylori (H. pylori) infection. Quantification of the number of GFP-LC3 puncta in AGS and GES-1 cells presented as mean ± SD, ^aP < 0.05 control vs H. pylori infection; B: The autophagosomes and autophagolysosomes at 24 h after H. pylori infection assayed by transmission emission microscopy (TEM), shown are a typical autophagosome (black arrowheads) and autophagolysosome (white arrowheads). Quantification of GFP-LC3 puncta in AGS and GES-1 cells shown as mean ± SD, ^aP < 0.05 vs control; C: The protein levels of light chain (LC)3B-I and LC3B-II at 12 h and 24 h after infection with H. pylori analyzed by western blot; D: Analysis of the expression of mir-30d at 12 h and 24 h after infection with H. pylori in both cell lines by quantitative polymerase chain reaction (q-PCR). Results shown as mean ± SD, ^aP < 0.05 vs control.

Figure 2

Mir-30d mimic represses autophagy in response to Helicobacter pylori infection in AGS and GES-1 cell lines. A: mir-30d expression in AGS and GES-1 cells with or without H. pylori infection at 48 h after mir-30d mimic transfection. Results shown as mean ± SD, ^aP < 0.05 vs control; B: The protein levels of LC3B-I and LC3B-II in mir-30d mimic transfected AGS and GES-1 cells with or without 24 h H. pylori infection; C: GFP-LC3 puncta in mir-30d transfected AGS and GES-1 cells with or without H. pylori infection (Results shown as mean ± SD, ^aP < 0.05 vs control mimic transfected cells); D: Quantification of autophagosome and autophagolysosome in mir-30d mimic transfected AGS and GES-1 cells with or without 24 h H. pylori infection (results shown as mean ± SD, ^aP < 0.05 vs control mimic transfected cells). H. pylori: Helicobacter pylori.

Figure 3

Mir-30d inhibitor upregulates autophagy in response to Helicobacter pylori infection in AGS and GES-1 cell lines. A: mir-30d expression in AGS and GES-1 cells with or without H. pylori infection at 48 h after mir-30d inhibitor transfection. ^aP < 0.05, with mir-30d inhibitor vs without mir-30d inhibitor; B: LC3B-I and LC3B-II protein levels in AGS and GES-1 cells with or without 24 h H. pylori infection; C: GFP-LC3 puncta in mir-30d transfected AGS and GES-1 cells with or without H. pylori infection (results shown as mean ± SD, ^aP < 0.05 vs control oligos transfected cells); D: Autophagosome and autophagolysosome in mir-30d transfected AGS and GES-1 cells with or without H. pylori infection (results shown as mean ± SD, ^aP < 0.05 vs control oligos transfected cells). H. pylori: Helicobacter pylori.

Figure 4

Multiple core proteins in the autophagy pathway are direct targets of mir-30d in gastric epithelial cells. A: Luciferase reporter assay with plasmids bearing wild type or mutant 3′UTR binding sites of mir-30d in AGS cells with mir-30d mimic or control oligos. Luciferase activity of mir-30d mimic transfected cells was normalized to control mimic transfected cells. Results are shown as mean ± SD, ^aP < 0.05 vs control; B: mRNA levels of autophagy related (ATG)2B, ATG5, ATG12, beclin 1 (BECN1), and BNip3-like protein (BNIP3L) in both cell lines with mir-30d mimic or control mimic transfection. Results are shown as mean ± SD, ^aP < 0.05 vs control; C: ATG2B, ATG5, ATG12, BECN1, and BNIP3L protein levels in both cell lines with mir-30d mimic or control mimic transfection; D: The mRNA levels of ATG2B, ATG5, ATG12, BECN1, and BNIP3L in two cell lines with or without mir-30d inhibitor transfection. Results are shown as mean ± SD, ^aP < 0.05 vs control.

Figure 5

Mir-30d increases intracellular survival of Helicobacter pylori in AGS cells. A: Gentamicin protection assay for the number of colony forming units (CFU) of H. pylori during a 60 h H. pylori infection with different treatment; B: The results from 24 h H. pylori infection, results are shown as mean ± SD, ^aP < 0.05 vs control. H. pylori: Helicobacter pylori.