Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion

Abstract

Unexplained recurrent spontaneous abortion (URSA) is a serious reproductive issue that affects women of childbearing age. Studies have shown a close association between disrupted circadian rhythm and impaired epithelial-mesenchymal transition (EMT) process in trophoblasts during URSA, though the underlying mechanism has not been elaborated. This study aims to investigate the regulatory relationship between circadian rhythm gene cryptochrome 2 (CRY2) and ferroptosis on the migratory ability of trophoblast cells. Cell proliferation experiments, wound healing assays, and expression of related markers were conducted to verify the EMT efforts. Trophoblastic ferroptosis was confirmed by the expressions of malondialdehyde, glutathione, mitochondrial membrane potential, divalent iron ions, and expression level of related genes. The results showed significant increased expression of CRY2 and decreased expression of brain and muscle ARNT-like protein 1 (BMAL1) in the URSA villous tissues, accompanied by iron-dependent oxidative changes and abnormal expression of ferroptosis related proteins. It was observed that CRY2 and BMAL1 was co-localized and function as a feedback loop, which regulated the dynamic changes of EMT-related markers in trophoblast cells. CRY2 promoted trophoblastic ferroptosis, while BMAL1 had the opposite effect. Particularly, the ferroptosis inhibitor (Ferrostatin-1) effectively reversed the trophoblastic ferroptosis and EMT inhibition caused by CRY2 overexpression. Collectively, these results suggest that CRY2 regulates trophoblastic ferroptosis and hinders cellular EMT and migratory ability by suppressing BMAL1 expression.