Journal: Cancer & Metabolism
Article Title: FOXO3a/miR-4259-driven LDHA expression as a key mechanism of gemcitabine sensitivity in pancreatic ductal adenocarcinoma
doi: 10.1186/s40170-025-00377-3
Figure Lengend Snippet: miR-4259 targets the LDHA 3’UTR and inhibits LDHA-mediated gemcitabine resistance in PDAC. ( A ) The miR-4259 expression (left) and LDHA -3’UTR luciferase activity (right) in PANC-1 and PANC-1/GEM cells was measured by RT-qPCR and a luciferase reporter assay, respectively. The RT-qPCR data were normalized to the level of U47 RNA in each individual sample. ( B ) A schematic diagram representing the predicted miR-4259-binding sequences or the mutated versions of the miRNA (left). The luciferase reporter activity (right) of the LDHA -3’UTR wild-type (+ 1 ~ + 937) and LDHA -3’UTR mutant reporters (mutant sites: 498, 498/518 and 498/518/818) were measured by a dual-luciferase reporter assay in HEK-293T cells transfected with miR-4259 and a reporter at different ratios. ( C ) The luciferase reporter activity of the LDHA -3’UTR wild-type and LDHA -3’UTR mutant reporters (triple-mutant sites, 498/518/818) in PANC-1/GEM and SUIT-2 cells and their expression of miR-4259. ( D ) The LDHA and miR-4259 expression (left) of PANC-1/GEM cells transfected with the indicated plasmids were analyzed by Western blotting and RT-qPCR, respectively. The cell viability (right) of these transfectants in the presence of gemcitabine treatment was measured by the MTT assay. ( E ) The LDHA and miR-4259 expression (left) of PANC-1 cells were analyzed by Western blotting and RT-qPCR, respectively. The cell viability ( right ) of these transfectants in the presence of gemcitabine treatment was measured by the MTT assay. The results are presented as the means ± s.e.m. of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 and n.s. not significant (two-tailed Student’s t -test)
Article Snippet: Five micrograms of total RNA obtained from PANC-1 and PANC-1/GEM cells were labeled and hybridized on miRNA microarrays (using the Human miRNA OneArray ® v2 (Phalanx Biotech Group, San Diego, CA, USA).
Techniques: Expressing, Luciferase, Activity Assay, Quantitative RT-PCR, Reporter Assay, Binding Assay, Mutagenesis, Transfection, Western Blot, MTT Assay, Two Tailed Test
