Figure 3
Helicobacter pylori -induced downregulation of mature miR-371, miR-372 and miR-373 levels in AGS cells. (A) Micro RNAs (miRNAs) content of AGS cells cocultured with H. pylori was determined by high-throughput '454' pyrosequencing. The graph represents the miRNAs content in reads of uninfected AGS cells (y axis) versus infected cells (x axis). miR-372, miR-373 and miR-371-5p are shown (empty plots). The trend line is represented, R2 = 0.9556 [see Additional File 1, Table S1 for total number of reads of each miRNA and Fisher's exact test P values]. (B) Endogenous expression of mature miR-371-3p, miR-372 and miR-373 (black bars), or miR-19b, miR-106b, miR-21 and miR-93 (grey bars), was determined by quantitative real time PCR (RT-qPCR) in AGS cells 24 h post infection at multiplicity of infection (MOI) 100. Bars indicate the relative miRNA expression normalized to SNORD49A (RNU49) and compared to uninfected AGS cells. Data represent mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001. (C) Northern blot analysis of mature miR-372, miR-373 and pre-miR-373 expression in uninfected (lane 2) or H. pylori-infected AGS cells (lane 3) in the same conditions as in (A). lane 1: M, size marker (nucleotides). A representative blot is shown. (D,E) Expression of the primary miR-371-372-373 cluster transcript after different time of infection (MOI 100) (D), and different MOI (6 h post infection) (E). Bars indicate the relative primary polycistronic miRNA transcript (pri-miRNA) expression determined by RT-qPCR, normalized to the ribosomal protein P0 mRNA and compared to uninfected AGS cells. A representative experiment is shown.