Research Paper Volume 9, Issue 8 pp 1885—1897

Epigenetic silencing of miR-338 facilitates glioblastoma progression by de-repressing the pyruvate kinase M2-β-catenin axis

Figure 2. MiR-338 binds to and degrades PKM2 transcripts through the RISC. (A) Graphic of the seed sequence of miR-338 matched with the 3′-UTR of the PKM2 gene, and the design of wild-type or mutant PKM2 3′-UTRs containing reporter constructs. Luciferase reporter assays in glioma cells after co-transfection of cells with wild-type or mutant PKM2 3′-UTRs and miRNA. The data represent the fold-change in the expression (mean and standard error) of three replicates (P<0.05). (B) Western blot of AGO2 protein immunoprecipitated from cell extracts with an AGO2 antibody, or IgG. The amount of PKM2 bound to AGO2 or IgG was measured by qrt-PCR in the presence of miR-338 mimics or miR-Scr (P<0.05). (C) Western blot of the effect of miR-338 overexpression on PKM2 protein expression after cells were transfected with the PKM2 plasmid or plain vector. (D) Western blot of PKM2 expression 48 hours after cells were transfected with miR-Scr/miR-338 or with inhibitor-NC/miR-338 inhibitor. (E) Qrt-PCR of PKM2 mRNA expression 48 hours after transfection (P<0.01).