Research Paper Volume 11, Issue 22 pp 10016—10030

Silencing of long non-coding RNA H19 downregulates CTCF to protect against atherosclerosis by upregulating PKD1 expression in ApoE knockout mice

Figure 4. H19 is capable of suppressing the transcriptional level of PKD1 through binding to CTCF. (A) The binding ability of H19 to CTCF assessed by RIP. *, p < 0.05 vs. IgG. (B) The predicted binding site of CTCF on the promoter region of PKD1 through JASPAR (http://jaspar.genereg.net/). (C) The luciferase activity of PKD1-Wt/Mut after transfection of oe-CTCF or CTCF-ASO determined by dual-luciferase reporter gene assay. * p < 0.05 vs. the oe-NC group; # p < 0.05 vs. the NC-ASO group. (D) The binding ability of CTCF to the promoter region of PKD1 evaluated by ChIP assay. * p < 0.05 vs. IgG. (E) The transfection efficiency of oe-H19 and sh-CTCF determined by RT-qPCR. * p < 0.05 vs. the oe-H19 + sh-NC group. (F, G) The mRNA and protein level of PKD1 normalized to GAPDH after overexpressing H19 or knocking down CTCF measured by RT-qPCR and Western blot analysis (the unprocessed blots are shown in Supplementary Figure 2). * p < 0.05 vs. the oe-H19 + sh-NC group. The data were measurement data and expressed by mean ± standard deviation. Data differences between two groups were analyzed by unpaired t-test; comparisons made among multiple groups were analyzed by one-way ANOVA. n = 6. The experiments were repeated three times independently.