Fig. 4
FUS promotes biogenesis of circROBO1 and KLF5 enhances transcription of FUS. a According to prediction of CircInteractome, an online database (https://circinteractome.nia.nih.gov/), the flanking intron downstream of circROBO1 harbored binding sites of FUS. b FUS was upregulated in BC according to the TCGA database and upregulated in I-IV stage of BC. c The Pearson correlation analysis showed that the mRNA expression of circROBO1 had a positive correlation with FUS in human BC tissues. d Knockdown of FUS or overexpression of FUS significantly decreased or increased the expression of circROBO1 in BC cells. e The results of RNA pull-down assays analyzed by western blot. f Schematic illustration of truncated pre-ROBO1 biotin-labeled probes used in RNA pull-down assays and the results of RNA pull-down assays were analyzed by western blot. g RIP assay was conducted using BT-549 lysates and FUS antibodies. Enrichment of pre-ROBO1 was analyzed by RT-qPCR. h Schematic diagram illustrated the structure of back splicing formation verification vector pc-HA-ROBO1 by inserting a HA tag in the 5’ side of exon 5 of circROBO1 to discriminate the internal circROBO1 with its downstream and upstream flanking 1000 nucleotide introns into the pcDNA3.1+ vector. The results of RT-qPCR by divergent primers crossed HA tag were displayed. i The Pearson correlation analysis showed that the mRNA expression of KLF5 had a positive correlation with FUS in human BC tissues. j Knockdown or overexpression of KLF5 downregulated or upregulated mRNA level of FUS analyzed by RT-qPCR. k and l JASPAR database indicated the potential KLF5 binding sites on the promotors of FUS. ChIP assays were conducted to verify the enrichment of the potential binding sites by KLF5 antibody in BT-549 cells. The ChIP results were analyzed by qPCR and nucleic acid electrophoresis. The dual-luciferase reporter assays were performed to validate the interaction between KLF5 and the first binding site (E1). The data are showed as the mean ± SD, *P < 0.05 **P < 0.01, ***P < 0.001 and all above experiments have been repeated for three times