Silencing circRNA-ZFAND6 induces trophoblast apoptosis by activating the mitochondrial pathway through the miR-575/SOD2 axis in unexplained recurrent spontaneous abortion

Abstract

Background: Unexplained recurrent spontaneous abortion (URSA) is a major problem in the field of human reproductive health. At present, several circRNAs have been reported to be differentially expressed and play an important biological function in pregnancy-related diseases. However, the role of circRNAs in URSA remains unclear.

Methods: Levels of circRNA and miRNA were examined by RT-qPCR. The si-RNA and overexpression plasmid were respectively used to silence and overexpress circRNA-ZFAND6. We investigated the biological function of circRNA-ZFAND6 on trophoblasts through CCK8, EdU, Flow cytometric assay, Wound-healing assays and Transwell. Dual luciferase activity assay was conducted to identify the interaction between miR-575 and circRNA-ZFAND6.

Results: We confirmed that circRNA-ZFAND6 was a stable circular RNA and was mostly localized in the cytoplasm. CircRNA-ZFAND6 was downregulated in placental villous tissues of URSA. CCK-8 and EdU assays showed that circRNA-ZFAND6 promoted the proliferation of HTR-8/SVneo cells. Flow cytometry and western blot assays prompted that circRNA-ZFAND6 obviously reduced cells apoptosis. Scratch wound healing and transwell assays revealed that circRNA-ZFAND6 had no effect on cell migration and invasion. CircRNA-ZFAND6 worked by adsorbing miR-575 through the ceRNA mechanism. MiR-575 can inhibit the proliferation and promote the apoptosis of HTR8/SVneo cells. SOD2 was identified as a direct target of miR-575 and was associated with mitochondrial apoptosis. Transmission electron microscopy, TMRM and ROS staining assays both suggested that circRNA-ZFAND6 affected mitochondrial apoptosis. Excessive trophoblast apoptosis was a key event to promote the development of URSA.

Conclusion: CircRNA-ZFAND6, which is low expressed in URSA and regulates the apoptosis of trophoblast cells, may affect the expression of SOD2 and thus affect mitochondrial apoptosis by regulating miR-575. This is closely related to the occurrence of URSA.

Keywords: CircRNA-ZFAND6; Hsa_miR_575; Pregnancy loss; SOD2; URSA.

Fig. 1

CircRNA-ZFAND6 is downregulated in placental villous tissues of URSA patients. (A) RT-qPCR analysis of the expression of hsa_circ_0000643, hsa_circ_0005142, hsa_circ_0002346, in 23 paired villi tissues from normal pregnant (Ctrl) and URSA groups. (B) Schematic diagram exhibited the formation of hsa_circ_0000643. (C) Sanger sequencing of the RT-PCR products of hsa_circ_0000643. The black arrow indicated the splicing site of hsa_circ_0000643. (D) RT-qPCR products using divergent and convergent primers showed the circularization of hsa_circ_0000643. GAPDH was used as a negative control. (E) The levels of circular and linear ZFAND6 were detected by using RT-qPCR in HTR8/SVneo cells treated with RNase R. (F) RT-qPCRanalysis of the hsa_circ_0000643 and ZFAND6 expression in HTR8/SVneo cells under the treatment with actinomycin D. (G) RNA FISH detected hsa_circ_0000643’s subcellular localization in HTR8/SVneo cells. Nuclei was stained with DAPI. Scale bar, 20 μm. (H) Clinical characteristics of the patients. (I) Expression of hsa_circ_ZFAND6 in 4 groups of villus tissues of NP group (Ctrl), first missed abortion group (MA) and URSA group. Three different independent experiments with three technical repetitions were performed. Data were expressed as the mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ns, not significant

Fig. 2

Silencing circRNA-ZFAND6 inhibits the proliferation and promotes the apoptosis of HTR8/SVneo cells (A, B) Effects of circRNA-ZFAND6 knockdown or overexpression on cell proliferation were monitored via CCK-8 and EdU assays. Scale bar, 50 μm. (C) The apoptotic rate was detected through flow cytometry transfected with si-circZFAND6 or circZFAND6. (D) The migration ability was assessed by scratch wound healing assay. Magnification, x40. (E) Transwell invasion and migration assay in transfected HTR8/SVneo cells. Scale bar, 100 μm. (F) Effects of circRNA-ZFAND6 silencing or overexpression on the levels of apoptosis-related proteinswere measured via western blot assay. GAPDH served as the internal control. Three different independent experiments with three technical repetitions were performed. Data were expressed as the mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ns, not significant

Fig. 3

CircRNA-ZFAND6 serves as a molecular sponge for miR-575and functions of miR-575in HTR8/SVneo cells. (A) The level of miR-575 in villus tissues of Ctrl and URSA (n=23) were measured via qRT-PCR. (B) RT-qPCR was performed to evaluate the transfection efficiency of miR-575 mimics and miR-575 inhibitor compared with their related control. (C) Schematic representation of miR-575 binding sites on circRNA-ZFAND6. The red part was the mutated sequences of circRNA-ZFAND6. Dual-luciferase reporter assay was performed in HEK-293T cells after transfection. (D, E) EdU and CCK8 analysis of the cell proliferation ability after transfected with miR-575 mimics and miR-575 inhibitor. Scale bar, 50 μm. (F, H) Flow cytometric analysis of cell apoptosis in treated HTR8/SVneo cells. (G) Western blot analysis of the protein expression level of Bax, Bcl-2, caspase9 and cleaved-caspase3 after transfected in HTR8/SVneo cells. GAPDH served as the internal control. Three different independent experiments with three technical repetitions were performed. Data were expressed as the mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ns, not significant

Fig. 4

SOD2 is a direct target of miR-575. (A) Schematic representation of miR-575 binding sites on SOD2. The red part was the mutated sequences of SOD2. Dual-luciferase reporter assay was performed in HEK-293T cells after transfection. (B) RT-qPCR analysis of the mRNA expression level of SOD2 in HTR-8/SVneo cells transfected with si-circZFAND6 and in villus tissues of Ctrl and URSA (n=23). (C) Western blot analysis of SOD2 protein level in transfected HTR-8/SVneo cells. (D) Electron microscope images showed the morphological changes of mitochondriain HTR-8/SVneo cells after silencing circRNA-ZFAND6. Scale bar, 1 μm. (E, F) Flow cytometry and fluorescence microscopy were used to analyze the changes of mitochondrial membrane potential. (G) Fluorescence microscopy was used to analyze the changes of intracellular ROS after transfected with si-circZFAND6. Scale bar, 20 μm. Three different independent experiments with three technical repetitions were performed. Data were expressed as the mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ns, not significant

Fig. 5

MiR-575 rescues the effect of circRNA-ZFAND6 in HTR8/SVneo cells. (A-C) Effects of circRNA-ZFAND6 knockdown on cell proliferation and apoptosis were abrogated by miR-575 inhibitor. Scale bar, 50 μm. (D) Western blotting demonstrated the rescue ability of miR-575 inhibitor on circRNA-ZFAND6 knockdown and the rescue abilityof miR-575 mimics on circRNA-ZFAND6 overexpression in HTR8/SVneo cells. Protein levels of Bax, Bcl-2, caspase9, cleaved-caspase3 were determined. GAPDH served as the internal control. (E) Flow cytometry analysis showed that miR-575 inhibitor rescued the change of mitochondrial membrane potential caused by si-circZFAND6. Three different independent experiments with three technical repetitions were performed. Data were expressed as the mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ns, not significant. MiR-575 inhibitor rescues the effect of circRNA-ZFAND6 silencing in HTR8/SVneo cells

Fig. 6

The placental trophoblast apoptosis of URSA increased significantly. (A) Western blot analysis of the expression level of apoptosis-related proteins in villus tissues of Ctrl and URSA group (n=3). GAPDH served as the internal control. (B) RT-qPCR assay was performed to evaluate the mRNA expression of apoptosis-related genesin villus tissues of Ctrl and URSA group (n=3). (C) Tunel staining of villus tissues from Ctrl and URSA group (n=3). (D) SOD2 protein expression was detected by immunohistochemistry (IHC) in Ctrl and URSA villus tissues (n=3). Representative images of SOD2 expression detected by IHC assays were shown. (E) Western blot analysis of the protein expression level of SOD2 in 3 groups of villus tissues of Ctrl, MA and URSA group. Three different independent experiments with three technical repetitions were performed. Data were expressed as the mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, ns, not significant