Skip to main content
Fig. 5 | Molecular Cancer

Fig. 5

From: m6A-dependent glycolysis enhances colorectal cancer progression

Fig. 5

METTL3 regulates HK2 and SLC2A1 (GLUT1) mRNA levels and stability depending on its m6A methyltransferase activity. (a-b) Real-time PCR and Western blot assays were performed to analyze the relative HK2 levels and SLC2A1 (GLUT1) in HCT116 cells after knockout (a) or knockdown (b) of METTL3 in HCT116 cells, n = 3, nonparametric Mann–Whitney test. (c) Real-time PCR and Western blot assays were performed to analyze the relative HK2 levels and SLC2A1 (GLUT1) in DLD-1 cells after transfection with pcDNA3.1, pcDNA3.1-METTL3 and pcDNA3.1-METTL3-mut, n = 3, nonparametric Mann–Whitney test. (d-e) Hexokinase activity was measured by colorimetric analysis after knockout (d) or knockdown (e) of METTL3 in HCT116 cells, n = 3, nonparametric Mann–Whitney test. (f) Hexokinase activity was measured by colorimetric analysis after transfection with pcDNA3.1, pcDNA3.1-METTL3 and pcDNA3.1-METTL3-mut in DLD1 cells, n = 3, nonparametric Mann–Whitney test. (g-i) Luciferase activity was measured in HCT116 cells transfected with control siRNA, METTL3 siRNA, pcDNA3.1, pcDNA3.1-METTL3 and pcDNA3.1-METTL3-mut. The luciferase reporters expressing WT or mutant human HK2 5’UTRs (g) and WT or mutant human HK2 3’UTRs (h) and WT or mutant human SLC2A1 (GLUT1) 3’UTRs (i) were used. The results were showed in the form of relative firefly luciferase activity normalized to Renilla luciferase activity. n = 4, nonparametric Mann–Whitney test. (j-k) The HK2 and SLC2A1 (GLUT1) mRNA half-life (t1/2) was detected by real-time PCR in HCT116 cells transfected with control siRNA or METTL3 siRNA1/2, n = 3, nonparametric Mann–Whitney test. (l) The HK2 mRNA half-life (t1/2) was detected by real-time PCR in HCT116 cells transfected with control siRNA or IGF2BP2 siRNA1/2, n = 3, nonparametric Mann–Whitney test. (m-n) The SLC2A1 (GLUT1) mRNA half-life (t1/2) was detected by real-time PCR in HCT116 cells transfected with control siRNA, IGF2BP2 or IGF2BP3 siRNA1/2, n = 3, nonparametric Mann–Whitney test. (o) Agarose electrophoresis and real-time PCR analysis of RIP assays in CRC cells showing the direct binding between the IGF2BP2 protein with HK2 5’UTR and HK2 3’UTR, and as well as the direct binding between IGF2BP2/3 with SLC2A1 (GLUT1) 3’UTR. (p) RIP-qPCR revealed the binding enrichment of IGF2BP2 to HK2 5’UTR and HK2 3’UTR, as well as the binding enrichment of IGF2BP2/3 to SLC2A1 (GLUT1) HK2 3’UTR, were decreased following knockout of METTL3 in HCT116 cells, n = 3, nonparametric Mann–Whitney test

Back to article page