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

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.