Fig. 6

GBAP1 acts as the molecular sponge of miR-22-3p in HCC cells. A The upregulated genes by METTL3 based on RNA-seq data were subjected to Gene Ontology (GO) biological process analysis. The top 10 categories were shown. B and C Fractionation of nuclear and cytoplasmic RNA assay was applied to identify the subcellular localization of GBAP1 in Hep3B and MHCC97H cells. D Data overlap from three bioinformatics tools (Targetscan, microRNA.org and starBase) revealed that there were three potential miRNAs which could bind to GBAP1-3’UTR. And miR-22-3p was one of them. E and F RT-qPCR was applied to test the expressions of the potential miRNAs in Hep3B-GBAP1 subclones or MHCC97H-shGBAP1 subclone and the control subclones. ***P < 0.001 (Student’s t test). G The potential bind site between wild type of GBAP1 (GBAP1-WT) and miR-22-3p was identified by bioinformatics tools. And the mutant type of GBAP1 (GBAP1-MUT) was established. Double luciferase reporter gene assay revealed that miR-22-3p mimics repressed the fluorescence activity of GBAP1-3’UTR-WT, while miR-22-3p inhibitors enhanced the fluorescence activity of GBAP1-3’UTR-WT. The alteration of miR-22-3p expression had no effect on GBAP1-3’UTR-MUT. H and I RIP assay using antibody against Ago2 for pull-down showed that in Hep3B and MHCC97H cells both GBAP1 and miR-22-3p were enriched by Ago2 antibody. ***P < 0.001 (Student’s t test). J RT-qPCR was performed to analyze GBAP1 expression in HCC cell lines and normal hepatic cell LO2. ***P < 0.001 versus LO2 (two-way ANOVA). K The expression of miR-22-3p was explored by RT-qPCR in HCC tissues (n = 85) and non-tumor tissues (n = 85). ***P < 0.001 (Student’s t test). L Pearson correlation analysis showed that there existed a negative correlation between GBAP1 and miR-22-3p in HCC tissues (n = 85)