Trophoblast KHSRP deficiency induces DNA damage and apoptosis via TFPI2 in unexplained spontaneous miscarriage

INTRODUCTION: Trophoblast dysfunction and abnormal cell death are associated with unexplained spontaneous miscarriage (USM), while the underlying mechanisms remain unclear. This research aims to elucidate the pathogenic functions of the RNA-binding protein KH-type splicing regulatory protein (KHSRP) in USM. METHODS: We first identified reduced KHSRP expression in USM villi by performing integrative analysis of our and public RNA-seq data, followed by validation in clinical samples via RT-qPCR, western blotting, and immunohistochemistry. Functional studies in HTR-8/SVneo and JEG-3 cells with siRNA knockdown and plasmid overexpression assessed proliferation, apoptosis, cell-cycle progression, and DNA damage through CCK-8, EdU, flow cytometry, and immunofluorescence. Downstream targets of KHSRP were identified by integrating siKHSRP RNA-seq, villi RNA-seq, and RIP-seq data, and subsequently validated at both the transcript and protein levels. In vivo, placental Khsrp knockdown was achieved via intrauterine injection of self-complementary adeno-associated virus 8 carrying a short hairpin RNA (shRNA) targeting the Khsrp gene (scAAV-shKhsrp). RESULTS: KHSRP was downregulated in USM villi. In trophoblast cells, KHSRP loss impaired proliferation, caused G2/M arrest, and triggered DNA damage accumulation and apoptosis in a p53-independent manner. Mechanistically, KHSRP bound and destabilized tissue factor pathway inhibitors 2 (TFPI2) mRNA. KHSRP deficiency upregulated TFPI2, promoting DNA damage accumulation and apoptosis. In mice, intrauterine scAAV-shKhsrp injection induced embryo resorption, placental structural abnormalities, and increased TFPI2 expression. DISCUSSION: KHSRP is downregulated in USM villi, and its loss elevates TFPI2 by impairing mRNA decay, leading to DNA-damage accumulation and trophoblast apoptosis. Thus, KHSRP and TFPI2 may serve as potential therapeutic targets for USM.