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

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.

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