circFTO binding with IGF2BP2 regulates trophoblast cells proliferation, migration, and invasion while mediating m6A modification of CCAR1 mRNA in spontaneous abortion.

Abstract

Background: Spontaneous abortion (SA) is a complex reproductive disease that poses significant clinical challenge. Circular RNAs (circRNAs), a specific class of endogenous non-coding RNAs, hold significant potential for preclinical diagnosis and therapeutic interventions in various diseases. However, the precise roles of circRNAs in SA have yet to be fully elucidated.

Methods: In this study, we employed RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) to identify an upregulated circRNA, circFTO (hsa_circ_0005941), in the placental villi of SA patients. We conducted in vitro and in vivo experiments to ascertainthe functional significance of circFTO in trophoblast cell lines. The molecular mechanisms associated with circFTO were predicted using online databases and confirmed through RNA immunoprecipitation (RIP) assays, Western blotting, and rescue experiments.Additionally, Actinomycin D was employed to assess changes in the stability of target messenger RNA (mRNA) under different treatments. Furthermore, colorimetric examinations were used to evaluate the m6A methylation levels of trophoblast cells, and meRIP-qPCR assays confirmed the m6A modification of CCAR1 mRNA.

Results: Our findings revealed that circFTO was upregulated in the placenta of SA patients. Functionally, downregulating circFTO expression enhanced trophoblast cell proliferation, migration, and invasion. Conversely, overexpression of circFTO inhibited these functions in trophoblast cells. Trophoblast organoids derived from normal pregnancies exhibited reduced proliferation upon overexpression of circFTO. Bioinformatics prediction and subsequent experiments demonstrated that circFTO directly bound to and negatively regulated IGF2BP2. Reducing the level of IGF2BP2 partially restored the alterations in trophoblast function caused by circFTO knockdown. Colorimetric assay, RNA decay experiments, and meRIP-qRT-PCR analysis revealed that circFTO knockdown increased m6A methylation levels in CCAR1 mRNA, while circFTO overexpression decreased m6A methylation levels. This modification is known to play a crucial role in Zygotic gene activation.

Conclusions: Our study unveils the pivotal functions of circFTO within trophoblasts and elucidates a unique circFTO-IGF2BP2-CCAR1 axis, which may hold significant potential as diagnostic and therapeutic targets for the treatment of SA.

Clinical trial number: Not applicable.