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Fig. 2 | Molecular Cancer

Fig. 2

From: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

Fig. 2

CircPTK2 binds directly to miR-429/miR-200b-3p in NSCLC cells. a Schematic description for the subcloning of the predicted miR-429/miR-200b-3p binding sites of circPTK2 exon11 in psiCHECK-2 luciferase vector. Predicted duplex formation between miR-429/miR-200b-3p and the wild-type/mutant of miR-429/miR-200b-3p binding sites of circPTK2 exon11 was shown. The entire subcloning sequences were listed in Additional file 4: Table S2. b, c Relative luciferase activity of the wild-type/mutant circPTK2 exon11 reporter gene in A549 and H226 cells transfected with miR-429/miR-200b-3p or negative control (miR-NC). Scrambled sequence was used as miR-NC. Relative Renilla luciferase activity was determined after normalizing against the firefly luciferase activity. d According to the flowchart outlining the experimental procedures (left), anti-AGO2 RIP was conducted in A549 cells transiently overexpressing miR-429/miR-200b-3p or miR-NC, and followed by RT-PCR and gel-staining analyses to detect circPTK2 enrichment (right). The 10% input was obtained as positive control before immunoprecipitation, and subjected to RT-PCR to confirm the presence of circPTK2. Anti-IgG antibody was used as a negative control. Red arrows represent divergent primers spanning the back-splice junction site of circPTK2. e According to the RNA pull-down flowchart (left), whole-cell lysates from A549 cells were incubated with biotinylated probes against circPTK2; after pull-down, endogenous circPTK2 (middle) and miR-429/miR-200b-3p (right) enrichments were detected by qRT-PCR. Results were presented as the percentage of pull-down to input. PD, pull-down. f Co-localization between circPTK2 (red) and miR-429/miR-200b-3p (green) was observed (arrowheads) by fluorescence in situ hybridization in A549 cells. Cell nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. g CircPTK2 expression in A549 and H226 cells transiently overexpressing circPTK2. Cells were transiently transfected with pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector (upper panel) for 48 h or 72 h and then subjected to qRT-PCR analysis (bottom panel). The empty vector was served as negative control. In the upper panel, the subcloned sequence in quadrate box includes front circular frame, back circular frame of circRNA biogenesis (grey part) and full-length of 584-bp circPTK2 (red part). h, i Endogenous miR-429/miR-200b-3p levels in A549 and H226 cells transiently overexpressing circPTK2. *P < 0.05; **P < 0.01; ***P < 0.001

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