Figure 3. PTPN13 is targeted by miR-26a in NSCLC cells.

A. Schematic representation of predicted human miR-26a binding site at nucleotides 376−382 of the 3′ UTR of PTPN13 mRNA. The sequence of the mutated 3′ UTR in the corresponding site was also shown. B. qRT-PCR assays of PTPN13 expression in the indicated human NSCLC cell lines or immortalized bronchial epithelial cell line, BEAS-2B. Data are represented as the mean ± SD of three replicates. *P<0.05, ** P<0.01, vs BEAS-2B cells. C. qRT-PCR assays of PTPN13 expression in SPCA1 cells after transfection with control or miR-26a antagomirs. Data are represented as the mean ± SD of three replicates. ** P<0.01, vs control group. D. Western blot assays of PTPN13 expression in SPCA1 cells after transfection with control or miR-26a antagomirs. E. Western blot assay of PTPN13 expression in PC-9 cells after transfection with control or miR-26a mimics. F. Luciferase activity measured 24 h after co-transfection of HEK293 cells with a miR-26a mimic and a pGL3 construct containing wild-type or mutant PTPN13 3′UTR regions encompassing the predicted binding site of miR-26a as described in (A). Data were normalized to the luciferase activity of control (vehicle-transfected) cells, and are represented as the mean ± SD of three replicates. ** P<0.01, vs NC group. G. Western blot assays of PC-9 cells after transfection with control or PTPN13 targeted siRNAs. H. The IC50 values of gefitinib for PC-9 cells (a) transfected with PTPN13 siRNA (siPTPN13-2) were measured and plotted as described in Figure 2B and 2D. The IC50 values of gefitinib for SPCA1 cells (b) transfected with PTPN13 siRNA (siPTPN13-2) and/or miR-26a antagomirs. Data are represented as the mean ± SD of three replicates. *P<0.05, ** P<0.01, vs control group. In westrern blot, data are representative of 3 independent experiments. All the protein levels measured with densitometry and normalized to β-actin. Each bar represents the mean±SD from three experiments. **p<0.01vs control.