Fig. 4: miR-30d suppresses glycolysis by inhibiting downstream SLC2A1 and HK1 via directly targeting RUNX1.

Lactate production (a), glucose uptake (b) and ATP production (c) were measured by colorimetric analysis in two PDAC cells transfected with miR-30d mimics or miR-30d inhibitor. d mRNA expression levels of 34 aerobic glycolysis-related genes were evaluated using qRT–PCR in miR-30d mimics and miR-30d inhibitor transfected cell lines. e Protein expression levels of HK1, SLC2A1, and ENO1 were measured by western blot in two cell lines with miR-30d mimics and miR-30d inhibitor transfection. f Immunofluorescence staining of HK1 and SLC2A1 expression in Panc-1 cells with miR-30d mimics transfection. Scale Bars, up: 20 μm; down: 5 μm. g Schematic illustration of the protocol for screening transcription factors using PITA, TargetScan, miRDB, miRanda, and picTAR to predict miR-30d target genes and PROMO, QIAGEN, and CHIPBase to predict transcription factors targeting HK1 and SLC2A1. h mRNA expression levels of 5 transcriptional factors were evaluated using qRT–PCR in miR-30d mimics and miR-30d inhibitor transfected cell lines. i The correlation between miR-30d, RUNX1, SLC2A1 and HK1 expression of pancreatic tissues in the TCGA cohort. The square in the upper right corner demonstrates the Pearson correlation value between the indicated genes with blue indicating negative correlation and red indicating positive correlation. The square in the lower left corner shows the scatterplot matrix fitted trend line for indicated genes. **, correlation is significant at the 0.01 level; *, correlation is significant at the 0.05 level. j, k Protein expression levels of RUNX1 were measured by western blot and immunofluorescence staining in cells with indicated treatment. Scale bars = 20 μm. l Luciferase activity assays were performed to confirm the direct binding efficiency of miR-30d and its putative target RUNX1 with indicated treatment.