Fig. 3

Circ-MTHFD1L acts as a miRNA sponge for miR-615-3p. A RIP assay showed the association between AGO2 and circ-MTHFD1L. Top, IP efficiency of the anti-AGO2 antibody in Western blots. Bottom, relative enrichment represents RNA associated with AGO2 relative to the input control. An IgG antibody served as a control. B The potential target miRNAs of circ-MTHFD1L were predicted in the ENCORI, circInteractome, circbank, and circAtlas databases. C QRT-PCR was utilized to determine the relative expression levels of miR-516b-5p and miR-615-3p in precipitates from PANC-1-GR and BxPC-3-GR cell lysate pulled down by the circ-MTHFD1L probe or oligo probe. D Schematic diagram of the circ-MTHFD1L-WT and circ-MTHFD1L-Mut luciferase vectors. E Relative luciferase activities in PANC-1-GR and BxPC-3-GR cells co-transfected with circ-MTHFD1L-WT or circ-MTHFD1L-Mut miR-615-3p mimic, inhibitor, or corresponding negative control. F RIP assay was carried out with anti-AGO2 antibodies or IgG in PANC-1-GR cells after transfection with the miR-615-3p mimic or mimic NC, and qRT-PCR was then performed to detect the enrichment of circ-MTHFD1L and miR-615-3p. G The co-localization of circ-MTHFD1L (red) and miR-615-3p (green) was observed by fluorescence in situ hybridization (FISH) in PANC-1-GR cells. Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. H Volcano plots showing the up-regulated (red) and down-regulated (blue) miRNAs in PDAC from GSE112264, GSE113486 (fold change > 2 or < 0.5, P < 0.05). I The expression levels of miR-615-3p in PADC cells (8988, AsPC-1, BxPC-3, PANC-1, SW1990) and normal human pancreatic duct epithelial (HPDE) were analyzed by qRT-PCR. J The expression levels of circ-MTHFD1L in tumor and matched non-tumor tissues from 96 gemcitabine-treated PADC patients were analyzed by qRT-PCR. K Relative RNA level of circ-MTHFD1L in PFS < 10 months group and PFS ≥ 10 months group were analyzed by qRT-PCR. L The correlation between circ-MTHFD1L and miR-615-3p in 96 gemcitabine-treated PDAC patients was analyzed by Pearson correlation analysis. M-N The relative expression of miR-615-3p in PANC-1-GR or BxPC-3-GR cells was analyzed by qRT-PCR after indicated transfection. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, between the indicated groups