Fig. 5: MiR-22 controls EMT by suppressing Snail and MAPK1/Slug/vimentin feedback loop.

a Representative images of the western blotting assay. BCa cells were treated with 50 nM interfering RNA targeting human Slug mRNA (named siSlug) or siNC for 48 h. Knockdown of Slug inhibited vimentin protein expression in T24 and UM-UC-3 cells. b qRT-PCR assay. BCa cells were treated as (a). Knockdown of Slug inhibited vimentin mRNA expression in T24 and UM-UC-3 cells. c Statistical analysis revealed a positive correlation between the expression levels of slug and MAPK1 (left) or vimentin (right) mRNA in TCGA’s BCa cohort (n = 401). d Representative images of the western blotting assay. BCa cells were treated with 50 nM interfering RNA targeting human vimentin mRNA (named siVIM) or siNC for 48 h. Knockdown of vimentin suppressed phosphorylation of ERK2 (MAPK1), but did not affect expression levels of total ERK2 in T24 and UM-UC-3 cells. e Representative images of the western blotting assay. UM-UC-3 cells were transfected with lipo2000 only (NC), pc-DNA3.1- vimentin (VIM) or co-transfected VIM and miR-22 for 48 h. Overexpression of vimentin increased phosphorylation of ERK2 (MAPK1), but did not affect expression levels of total ERK2. MiR-22 mimic reversed the vimentin-induced ERK2 phosphorylation. f Representative images of the qRT-PCR assay. BCa cells were transfected with lipo2000 only (NC), VIM or siVIM for 48 h. Neither overexpression nor knocking down of vimentin affected the expression level of miR-22. g The 24-h transwell assay. BCa cells were transfected with lipo2000 only (NC), pc-DNA3.1- vimentin /MAPK1 (VIM/MAPK1) or co-transfected VIM/MAPK1 and miR-22. MiR-22 reversed migration and invasion that induced by MAPK1 (left) or Snail (right) overexpression in BCa cells. h A schematic diagram of the mechanisms under miR-22-mediated preoliferation, apoptosis and EMT regulation in BCa. Error bars represent the S.D. obtained from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001