Fig. 2: Metformin transcriptionally suppresses DHFR via E2F4.
From: Metformin sensitises hepatocarcinoma cells to methotrexate by targeting dihydrofolate reductase
A qRT-PCR analysis of DHFR mRNA levels in HepG2 cells treated with metformin (2.5 mM) for the indicated hours. B Diagram of the strategy for screening possible transcription factors (TFs) that regulate DHFR. TFs that might bind to the DHFR promoter (−1000 to 0) were collected from TF prediction websites, including GeneCards, hTFtarget and Consite. The common transcription factors that might bind to the DHFR promoter were further validated by the EPD motif tool. C qRT-PCR analysis of the mRNA levels of DHFR and E2F4, E2F6 or IRF1 in HepG2 cells expressing NTC shRNA or shRNAs against E2F4 (shE2F4), E2F6 (shE2F6) or IRF1 (shIRF1), respectively. D qRT-PCR and western blot analyses of DHFR expression in HepG2 cells expressing 3XFlag-EV or 3XFlag-E2F4. E Western blot analysis of the expression of E2F4 and DHFR in HepG2 cells treated with metformin (2.5 mM) for 48 h. F A diagram showing the potential E2F4 binding regions in the DHFR promoter. Regions 2 and 3 contain several potential E2F4 binding sites. Region 1 was used as the negative control. G A dual-luciferase assay was performed to identify E2F4 binding regions in the DHFR gene. Different regions containing predicted binding sites were inserted into a luciferase reporter vector. E2F4 was co-transfected with pGL3-Basic-Region 1 (R1), pGL3-Basic-Region 2 (R2) or pGL3-Basic-Region 3 (R3). H ChIP-qPCR analysis of E2F4 occupancy in the binding region in the DHFR gene in HepG2 cells using IgG or an anti-E2F4 antibody. I Luciferase assays were performed to determine the transcriptional activity of DHFR via E2F4 with or without metformin (2.5 mM) treatment. J qRT-PCR analysis of DHFR mRNA levels in HepG2 cells expressing NTC shRNA or shRNA against E2F4 (shE2F4) under metformin (2.5 mM) treatment for 24 h. K Western blot analysis of the expression of E2F4 and DHFR in HepG2 cells expressing NTC shRNA or shRNA against E2F4 under metformin (2.5 mM) treatment for 48 h. Band intensities for protein expressions in the western blot assay were quantitated by ImageJ and normalised to Actin. Data are presented as the mean (±SEM) or mean (±SD) values. Statistical significance was assessed by Student’s t-test or ANOVA followed by Dunnett’s or Tukey’s multiple comparisons test. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively, compared between the indicated groups. ‘ns’ indicates no significant difference between the indicated groups.
