Figure 4.

circRNA-DURSA regulates trophoblast apoptosis via the miR-760-HIST1H2BE axis

(A) HIST1H2BE mRNA and protein expression in HTR8/SVneo cells and primary human trophoblast cells transfected with si-circRNA-DURSA or si-circRNA-NC. (B) qRT-PCR and western blot analysis of HIST1H2BE mRNA expression and protein in HTR8/SVneo cells and primary human trophoblast cells transfected with miR-NC or miR-760 mimic. (C) Cell morphology by microscope. HTR8/SVneo cells were transfected with si-NC+inhibitor NC, si-circ-DURSA+inhibitor NC, or si-circ-DURSA+miR-760 inhibitor. The experiments of (C)–(H) were carried out in the three groups above. (D) Cell viability in treated HTR8/SVneo cells was assessed by CCK-8 assays. OD values were obtained at 24, 48, and 72 h after transfection. (E) Flow cytometric analysis of cell apoptosis in treated HTR8/SVneo cells and primary trophoblast cells of three groups. (F) Caspase-3 activity was measured to determine the activation level of the caspase-3 protein in the three groups. (G) HIST1H2BE mRNA expression levels were measured by qRT-PCR in treated HTR8/SVneo cells. (H) Western blot assays were performed to measure the H2B protein expression levels in treated HTR8/SVneo cells. (I) TargetScan database showing the putative binding sites of miR-760 related to HIST1H2BE. (J and K) HTR8/SVneo cells were co-transfected with psiCHECK-2-HIST1H2BE-3′ UTR WT or psiCHECK-2-HIST1H2BE-3′ UTR mutant and miR-760, or miR-NC. Luciferase activity was detected with luciferase reporter assays, normalized with Renilla activity. (L) HIST1H2BE mRNA expression in URSA and normal pregnancy placental villus (n = 22). (M and N) HIST1H2BE protein expression was detected by western blot and immunohistochemistry (IHC) in URSA and normal pregnancy placental villus (n = 22). Representative images of H2B expression detected by IHC assays are shown. Three different independent experiments with three technical repetitions were performed. Data are expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. NS, not significant.