Fig. 4

CircHIF1A/miR-361-5p/HIF1A forms a ceRNA network. A-E. Screening of target miRNAs and genes: (A) Cytoscape was used to generate the circRNA-miRNA-mRNA regulatory network of circHIF1A. (B) Target miRNAs were screened using bioinformatics methods. (C) Differential expression of miR-4677-3 and miR-361-5p in LIM1215 and LIM1215-R. (D) Differential expression of circHIF1A and HIF1A mRNA in LIM1215 and LIM1215-R. (E) Differential expression of HIF1α protein in LIM1215 and LIM1215-R. F-H. Validation of the binding relationship between miRNA-361-5p, circHIF1A and HIF1A: (F) RIP assay revealed significant enrichment of circHIF1A, miRNA-361-5p and HIF1A 3’UTR in the anti-AGO2 group compared to the IgG group. (G) LIM1215-R were transfected with biotin-labeled miRNA-361-5p-WT or miRNA-361-5p-Mut, and the levels of circHIF1A and HIF1A 3’UTR were analyzed by qRT-PCR. The relative ratio of IP to the input value is plotted. (H) Dual-luciferase reporter assays confirmed that miRNA-361-5p could bind both circHIF1A and HIF1A 3’UTR. I-K. CircHIF1A regulates downstream genes through HIF1A: (I) Comparison of the expression levels of GLUT1, LDHA, PFKFB3, PKM2 and HK2 mRNA and protein in LIM1215 and LIM1215-R. (J) Downregulation of circHIF1A in LIM1215-R resulted in altered expression of GLUT1, LDHA, PFKFB3, PKM2 and HK2 mRNA and protein. K. The promoter activity of GLUT1 and LDHA in LIM1215-R was higher than that in LIM1215. Downregulation of circHIF1A in LIM1215-R resulted in decreased promoter activity of GLUT1 and LDHA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001