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. 2022 Aug 1;12:928373. doi: 10.3389/fonc.2022.928373

Figure 4.

Figure 4

MiR-24-1 mechanistically activates FBP1 through enhancer. (A) The schematic diagram of pGL3-enhancer vector construction in dual luciferase reporter gene assay. The enhancer sequence containing miR-24-1 DNA locus located on 60 kb upstream from FBP1 was inserted into pGL3 vector to construct pGL3-enhancer vector. (B) The enhancer sequence containing miR-24-1 DNA locus can increase the activity of reporter gene. Co-transfection of miR-24-1 expression vector and pGL3-enhancer vector induced an increase of enhancer activity in dual luciferase reporter gene assay. (C, D) More enrichment of H3K27ac (C) and H3K4me1 (D) on miR-24-1 locus was observed by ChIP-qPCR after overexpressing miR-24-1. (E, F) The schematic diagram of CRISPR/Cas9 system. A deletion of 54bp in enhancer sequence containing miR-24-1 (E) was confirmed by Sanger sequencing (F). (G) The mRNA expression levels of FBP1 were detected by qPCR. FBP1 was increased when overexpressing miR-24-1, yet failed to be activated when enhancer was deleted. (G) Similarly, the protein expression levels of FBP1 were detected by western blot. Results are shown as mean ± S.D., ****p < 0.0001, ***p < 0.001, **p < 0.01, ns means not significant.