FIGURE 5.

MiR‐21‐3p regulates autophagy and chemoresistance by targeting PRKAA2. (A) Schematic diagram shows the putative‐binding site of miR‐21‐3p with PRKAA2 predicted by TargetScan. (B) Luciferase reporter assay showed that luciferase activity decreased obviously after co‐transfection of miR‐21‐3p with the constructed PRKAA2 3′UTR‐wt plasmid. (C and D) Western blotting analysis of the expression of PRKAA2 in AGS‐5FU and AGS cells transfected with miR mimic and anti‐miR. (E and G) Apoptotic assays of AGS‐5‐FU cells transfected with miR mimic or co‐transfected PRKAA2 overexpression vector upon 5‐FU exposure (25 μM 48 h). (F and H) Apoptotic assays of AGS cells transfected with anti miR or co‐transfected with sh‐PRKAA2 in AGS upon 5‐FU exposure (5 μM 48 h). (I and K) Western blot analysis of caspase3, c‐caspase 3, LC3 and p62 in AGS‐5FU transfected with miR mimic or co‐transfected with PRKAA2 overexpression vector upon 5‐FU exposure (25 μM 48 h). (J and L) Western blot analysis of caspase3, c‐caspase 3, LC3 and p62 in AGS transfected with anti‐miR or co‐transfected with sh‐PRKAA2 in AGS upon 5‐FU exposure (5 μM 48 h). (M) Immunofluorescence analysis (scale bar 20 μm) and transmission electron microscopy (TEM) images (scale bar = 2 μm or 0.5 μm) of AGS‐5FU transfected with miR mimic or co‐transfected PRKAA2 overexpression vector upon 5‐FU exposure (25 μM 48 h). Left lower panel: Quantification data of autolysosome and autophagosome in AGS‐5FU. Right lower panel: Quantification data of autophagic vacuoles (AV) counts in AGS‐5FU. (N) Immunofluorescence analysis (scale bar 20 μm) and TEM images (scale bar = 2 μm or 0.5 μm) of AGS transfected with anti‐miR or co‐transfected with sh‐PRKAA2 upon 5‐FU exposure (5 μM 48 h). Left lower panel: Quantification data of autolysosome and autophagosome in AGS. Right lower panel: Quantification data of AV counts in AGS. The number of AV of 15 cells was counted in each section. (Graph represents mean ± SD; *p < .05, **p < .01 and ***p < .001)