Figure 5.

miR-223 triggered by lactate targets Fbw7 and promoted DLBCL proliferation. (A) qPCR analysis of the relative miR-223 expression in SU-DHL-2 and OCI-LY-3 cell lines. Lactate was treated into cells with the indicated concentration gradient. The mean ± SD is shown for five independent experiments. **P < 0.01. ***P < 0.001; one-way ANOVA. (B) western blots showed miR-223 inhibitor increases Fbw7 expression and mainly reduces LDHA expression in Glycolysis relative proteins. And the expression of Fbw7, LDHA, HK2, GLUT1, PDK1 were shown. (C) miR-223 mimic promotes SU-DHL-2 cell proliferation in vivo. Representative images of tumors proliferation in NOD-SCID mice. Mice were implanted with 1 × 10⁷ cells into the flanks of mice (n = 5). Here, mice were treated with miR-223 agomir and agomir of normal control twice a week. 27 days after tumor implantation, mice were sacrificed and tumor volume was measured. 1 cm for Scale bar. A multi-way classification analysis of variance tests was performed to assess data obtained from the tumor volume assays and data are shown as mean ± SD. P < 0.01. (D) binding sites of miR-223 and the 3’-UTR of Fbw7 were shown for three conserved sites which was determined by the TargetScan algorithm. (E) reporter constructs containing a position of Fbw7 190-197 3’-UTR region (wild-type, WT) or Fbw7 (MUT) with mutated miR-223 binding site of were co-transfected with a control oligo (mimic ctrl) or miR-223 mimic oligo into HEK293 cells. Luciferase activity was detected 24 hours after transfection. Data are shown as mean ± SD; n=5. *P < 0.01; **P < 0.01; one-way ANOVA. (F) Fbw7 and miR-223 inhibitor reduces intracellular LDH in U2932 and SU-DHL-2 cells. The mean ± SD is shown for five independent experiments. **P < 0.01; one-way ANOVA. (G) Fbw7 and miR-223 inhibitor reduced ATP production in U2932 and SU-DHL-2 cells. The mean ± SD is shown for five independent experiments. **P < 0.01; one-way ANOVA.