Figure 7: miR-128-3p targets Axin1, SFRP2, WIF1, SMURF2 and PP1c in NSCLC cells.

(a) Targetscan tool showing schematic representation of putative binding sites for miR-128-3p in 3′-UTRs of Axin1, SFRP2, WIF1, SMURFS and PP1c. (b) WB analysis of the protein levels of Axin1, SFRP2, WIF1, SMURF2 and PP1c in the indicated cells. (c) By immunoprecipitation against Ago1, RIP analysis reveals the interaction of miR-128-3p with the 3′-UTRs of Axin1, SFRP2, WIF1, SMURF2 or PP1c mRNA to form miRNP complexes. IgG immunoprecipitation, as well as the interaction of miR-128-3p with GAPDH and 5s rRNA, were used as negative controls. (d) Luciferase assay of pGL3-Axin1-3′-UTR, pGL3-SFRP2-3′-UTR, pGL3-SMURF2-3′-UTR, pGL3-PP1c-3′-UTR or pGL3-WIF1-3′-UTR reporters in the indicated cells, co-transfected with increasing amounts (20 and 50 nM) of the indicated oligonucleotides. The sequence of the miR-128-3p mutant is shown. (e) Effects of restored expression of Axin1, SFRP2, WIF1, SMURFS and PP1c in miR-128-3p-overexpressing cells on luciferase activities of the TOP/FOP reporter and TGF-β reporter. (f) Effects of restored expression of miR-128-3p target genes on cell migration and self-renewal measured by Transwell migration assay and sphere formation assay, respectively, in the indicated NSCLC cells. Error bars represent mean±s.d. derived from three independent experiments. A two-tailed Student’s t-test was used for statistical analysis (*P<0.05, **P<0.01).