CircLphn3 functions as a sponge for miR-185-5p and upregulates tight junction genes.
(A, C) CircLphn3 and ZO-1, ZO-2, and occludin mRNA and protein expression were significantly upregulated after circLphn3 overexpression. (B) Representative western blot images of ZO-1, ZO-2, ZO-3, and occludin after circLphn3 overexpression. (D) The predicted binding sites (red bases) of miR-185-5p and circLphn3. (E) Relative luciferase activity in 293T cells transfected with the indicated plasmids. (F) miR-185-5p was strongly downregulated by circLphn3 overexpression. (G) The predicted binding sites of miR-185-5p and ZO-1 mRNA 3′UTR. (H) Dual luciferase reporter gene assay confirmed the binding sites of miR-185-5p and ZO-1 mRNA 3′UTR. (I) Representative western blot images of ZO-1, ZO-2, ZO-3, and occludin after miR-185-5p mimics transfection. (J) ZO-1 was significantly downregulated in bEnd.3 after miR-185-5p mimics transfection. All data are presented as mean ± SEM. The experiments were repeated five times. *P < 0.05. circLphn3: Circular Lphn3; Lphn3: latrophilin 3; mut: mutant; NC: negative control; ns: not significant; OE: overexpression; UTR: untranslated region; wt: wild type; ZO: zona occludens.