Fig 2: The miRNA-125-5p targets the 3′ UTR of VE-cadherin and is involved in mediating ERβ’s effect on VE-cadherin. (a) The qPCR detection of VE-cadherin mRNA at different time points after 10 nM E2 treatment. (b) Diagram of constructing the 3′ UTR of VE-cadherin (CDH5) using psiCHECK2 vector. (c) Knocking down ERβ (shERβ) decreased psiCHECK2-CDH5 luciferase activity. (d) The miRNA heatmap of clinical RCC samples (GSE37989) for screening key miRNAs targeting VE-cadherin (CDH5). Red stands for higher expression and blue stands for lower expression. (e) Algorithm for selecting potential tumor suppressor miRNA targeting VE-cadherin. (f) Binding site information of miR-125-5p with the 3′ UTR of VE-cadherin (CDH5) from Targetscan. (g) Correlation between miR-125b-5p and VE-cadherin expression in clinical ccRCC samples. (h–m) Interruption assays of miR-125b-5p in regulation of VE-cadherin (CDH5) and VM formation. Overexpression of miR-125b-5p in A498 cells interrupted sunitinib (STN)-induced VE-cadherin (CDH5) mRNA (h) and protein (i), and interrupted VM formation (l). Treating 786-O cells with the miR-125b-5p inhibitor (INH) rescued shERβ-decreased VE-cadherin (CDH5) mRNA (j) and VE-cad protein (k), and rescued VM formation (m). Scale bar, 20 μm. For (l,m), quantitations are on the right. Data is presented as mean ± SEM. ** p < 0.01, *** p < 0.001.

Fig 3: The circDGKD mediates the ERβ-regulated VE-cadherin and VM formation in RCC. (a) Schematic diagram of constructing shRNA plasmid of circDGKD targeting the junction region, and wild-type (Wt)/MRE-125 mutant (Mut) circDGKD overexpression plasmid by adding Alu element on the flank. (b) Knockdown of circDGKD (shcircDGKD) decreased VE-cadherin level in 786-O cells. (c) Overexpression of circDGKD increased VE-cadherin in A498 cells. (d) Knockdown of circDGKD and overexpression of miR-125b-5p both decreased psiCHECK2-CDH5 luciferase activity, whereas knockdown of circDGKD, on the basis of overexpressed miR-125b-5p, did not further decrease the luciferase activity. (e) Overexpression of Wt circDGKD, but not the circDGKD with MRE-125 binding site Mut, significantly increased the luciferase activity of psiCHECK2-CDH5, in the absence or presence of miR-125. (f–k) Interruption assays of ERβ and circDGKD regulation of VE-cadherin and VM formation. Overexpression of circDGKD restored shERβ-decreased VE-cadherin in both mRNA (f) and protein (g), and rescued the shERβ-impaired VM formation in 786-O cells (j). Sunitinib (STN) significantly increased VE-cadherin mRNA (h) and protein level (i) in A498 cells, and boosted the VM formation capacity (k), whereas this effect was interrupted by shcircDGKD. Scale bar, 20 μm. For (j,k), quantitations are at the right. Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001. NS: not statistically significant.

Fig 4: BUT induces specific tyrosine phosphorylation of VEC. (A) Phosphorylation of VEC at tyrosine-731, -685, and -658 (phospho-VECY731, Y685, and Y658) in response to various concentrations of BUT analyzed by western blotting. HAOECs were treated for 1 h with indicated concentrations of BUT. Phospho-VEC signals where normalized to the total VEC. Relative phospho-VEC signals were normalized to the level in control cells (dashed red line). ß-Actin served as a loading control. (B) Kinetic of BUT-induced phospho-VECY731 level analyzed by western blotting. Data display densiometric quantification of relative phospho-VEC and VEC signals normalized to the level in control cells (dashed red line) of n = 4 experiments. For better illustration, the identical dataset for 1 h treatment at 1 mM from panel (A) (phospho-VECY731) is shown. (C) Phospho-VECY731 level after 1 h treatment of HAOEC with BUT (1 mM) in the presence or absence of Ang-II (100 ng/mL). Data display densitometric quantification of relative phospho-signals as described in (B).

Fig 5: BUT engages Src kinases for specific tyrosine phosphorylation at VEC. (A) HAOECs were treated for 15 min before and during BUT incubation with PP2 (10 µM) or vehicle (DMSO). Subsequently, phospho-VECY731 levels were analyzed by western blotting. Phospho-signals of n = 4 experiments were quantified by densitometry. (B) Immune fluorescent microscopy of HAOECs treated with the vehicle (DMSO) or PP2 and BUT (5 mM) for 1 h and subsequently stained for VEC and general phospho-tyrosine residues. Scale bar (10 µm). (C) Validation of Src KD by RNAi. HAOECs were transfected with Src-specific siRNA or control-siRNA. Cells were lysed and analyzed for Src expression 12 h later by western blotting. (D) Stimulation of Src KD and control cells with BUT (1 mM) for 1 h and analysis of phosho-VECY731 expression as shown in Figure 2. Quantification corresponds to n = 6 experiments. (E) Phospho-SrcY416 western blotting of BUT-treated or -untreated cells, transfected with Ctrl or FFAR3 siRNA. Data display densiometric quantification of relative phospho-Src signals normalized to the level in BUT-untreated Ctrl siRNA-transfected cells (dashed red line) of n = 3 experiments.