Fig 2: IGF2BP3 decreases ZO-1 expression by synergizing with miR191-5p via the RISC. (A) ZO-1 mRNA expression was detected in the argonaute 2 complex using an RIP assay in HA22T cells transfected with pLKO and shIGF2BP3. (B) Reverse transcription-quantitative PCR was used to screen 6 potential miRNAs which may be able to regulate ZO-1 from the argonaute 2 complex in Huh-7 cells transfected with pWPI and IGF2BP3-cDNA. (C) Transwell invasion assays were performed using Huh-7 cells transfected with pWPI+pWPI, pWPI+oeIGF2BP3, oemiR-191-5p+pWPI or oemiR-191-5p+oeIGF2BP3. (D) Western blotting was performed to detect ZO-1 expression in Huh-7 cells transfected with pWPI+pWPI, pWPI+oeIGF2BP3, oemiR-191-5p+ pWPI or oemiR-191-5p+oeIGF2BP3. For C and D, the quantifications are shown on the right, and are presented as the mean ± SD. *P≤0.05 and **P≤0.01. ns, not significant; IGF2BP3, insulin-like growth factor II mRNA-binding protein 3; ZO-1, zonula occludens-1; miRNA/miR, microRNA; RISC, RNA-induced silencing complex; RIP, RNA interaction-precipitation; sh, short hairpin (RNA); oe, overexpression; NC, negative control.

Fig 3: Schematic diagram illustrating IGF2BP3 function. IGF2BP3 can decrease ZO-1 mRNA stability by enhancing miR191-5p expression and inducing the RISC-mRNA interaction. ZO-1, zonula occludens-1; IGF2BP3, insulin-like growth factor II mRNA-binding protein 3; RISC, RNA-induced silencing complex; miR, microRNA; RISC, RNA-induced silencing complex; UTR, untranslated region.

Fig 4: PDK4 is involved in m6A regulated cancer progression.The relative cell proliferation of wild type and Mettl3Mut/- HeLa cells stably transfected with vector control or PDK4 constructs; The relative cell proliferation of sh-control and sh-Mettl3 Huh7 cells stably transfected with vector control or PDK4 constructs; Wild type and Mettl3Mut/- HeLa cells stably transfected with vector control or PDK4 constructs were treated with increasing concentrations of Dox for 24 h, and the cell proliferation was tested. a The tumor growth curves of wild type and Mettl3Mut/- HeLa cells stably transfected with vector control or PDK4 constructs. b IHC (Mettl3 and PDK4)-stained paraffin-embedded sections obtained from wild type and Mettl3Mut/- HeLa cells. The scale bar is 50 μM. c Correlation between Mettl3 and PDK4 in cervical cancer patients (n = 169) from TCGA database. g–i The relative mRNA expression of Mettl3 (G), IGF2BP3 (H), or YTHDF1 in Oncomine datasets. j, k The Kaplan–Meier survival curves of DFS based on Mettl3 (j) or TBP (k) expression in cervical cancer patients from TCGA data base. l, m The Kaplan–Meier survival curves of DFS (l) or OS (m) based on PDK4 expression in cervical cancer patients from TCGA data base. Data are presented as means ± SD from three independent experiments. A representative from a total of three independent experiments is shown for (e). NS, no significant, by one-way ANOVA for (a), (b), and (d), by two-tailed unpaired Student’s t test for (f–i), and by two-sided log-ranjk test for (j–m).

Fig 5: IGF2BP3 improves miR191-5p-induced silencing complex function by binding the 3′-UTR of ZO-1 mRNA and promoting Ago2-mRNA interactions. (A) Sequence alignment of the ZO-1 3′UTR with wild-type vs. mutant potential miR191-5p targeting sites. The left red sequence represents the potential IGF2BP3 binding site of the ZO-1 mRNA 3′UTR. The right red sequence represents the potential miR191-5p binding site of the ZO-1 mRNA 3′UTR, and the crossed sequence represents deletion in the mutant ZO-1 3′UTR. (B) Luciferase reporter activity after transfection of wild-type and mutant ZO-1 3′UTR reporter constructs in HA22T cells with/without miR191-5p. (C) Sequence alignment of the ZO-1 3′UTR with wild-type vs. mutant potential IGF2BP3 targeting sites. The left red sequence represents the potential IGF2BP3 binding site of ZO-1 mRNA 3′UTR, and the crossed sequence represents deletion in the mutant ZO-1 3′UTR. The right red sequence represents the potential miR191-5p binding site of ZO-1 mRNA 3′UTR. (D) Luciferase reporter activity following transfection with wild-type or mutant ZO-1 3′UTR reporter constructs in Huh-7 cells with/without IGF2BP3-cDNA, and HA22T cells treated with/without IGF2BP3-shRNA, compared with the control cells. All quantifications are presented as the mean ± SD. *P≤0.05 and **P≤0.01. ns, not significant; IGF2BP3/IMP3, insulin-like growth factor II mRNA-binding protein 3; ZO-1, zonula occludens-1; UTR, untranslated region; miR, microRNA; sh, short hairpin (RNA); oe, overexpression; NC, negative control.