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. 2020 Apr 27;11(4):296. doi: 10.1038/s41419-020-2492-2

Fig. 4. DANCR is a critical downstream target of ZNF750 to regulate FOXC2 expression in a ceRNA manner.

Fig. 4

a Pathway enrichment analysis displayed that the differentially expressed genes (Fisher-exact test, p < 0.05) were enriched in pathways such as PI3K/Akt signaling, Hippo signaling, regulation of actin cytoskeleton and proteoglycans in cancer. b 64 genes as potential direct targets of ZNF750 occurring simultaneously in RNA-seq data and Boxer’s ChIP-sequencing data. c qRT-PCR showed the expression of DANCR in ZNF750 knockdown cells (left) and ZNF750-wt cells (right). GAPDH was performed as a loading control. Statistical analysis was performed with a two-sided t test. d Upper: A putative ZNF750 binding site on the -342 to -334 region before transcriptional start site of DANCR. Lower: ChIP-PCR showed that ZNF750 directly bound to the promoter of DANCR. e Dual luciferase assay showed that ZNF750 overexpression decreases the luciferase activity of DANCR reporter. This effect was abrogated when the ZNF750 binding site was mutated or deleted. P values were obtained using ANOVA. f DANCR localization in both cytoplasm and nuclear of KYSE150 cells as measured by RNA-FISH. DAPI labels the nucleus. Scale bars represent 100 μm. g The putative binding site of DANCR was found to be similar to the binding site of miR-4707-3p on 3’UTR of FOXC2. h Dual luciferase report assay showed miR-4707-3p mimics significantly reduced the luciferase activity of pSicheck2-DANCR-wt rather than pSicheck2-DANCR-mut. Statistical analysis was performed with a two-sided t test. (i) qRT-PCR showed the expression of DANCR in miR-4707-3p inhibition cells. j RIP showed DANCR and miR-4707-3p were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates. Statistical analysis was performed with a two-sided t test. k Dual luciferase assay showed that miR-4707-3p mimics decreased the luciferase activity of FOXC2-3’UTR reporter rather than mutant FOXC2-3′UTR reporter. P values were obtained using ANOVA. l, m Augmentation of miR-4707-3p led to a decrease of FOXC2 expression level whereas silence of miR-4707-3p caused an increase as measured by qRT-PCR (l) and western blot (m). Statistical analysis was performed with a two-sided t test. *p < 0.05, **p < 0.01,***p < 0.0001.