Downregulation of DNA ligases in trophoblasts contributes to recurrent pregnancy loss through inducing DNA damages

Abstract

Introduction: As key components of DNA repair pathways, DNA ligases catalyze the formation of phosphodiester bonds between DNA single strands, which function as a "glue" to seal the DNA breaks. DNA ligases play important roles in almost all the normal physiological processes for maintaining the stability of genomic DNA, but their functions in recurrent pregnancy loss (RPL) are still unclear.

Methods: Immunoblotting was used to determine protein level. DNA damages were examined by comet assay and cell viability was quantified by MTT assay. The cell apoptosis and cell cycle were examined by flow cytometry. The LIG4 mRNA degradation was quantified by qRT-PCR after actinomycin D treatment. The interactions between miRNAs and LIG4 were predicted by TargetScan and confirmed by dual luciferase assay.

Results: LIG1 and LIG4 were downregulated in RPL patients, while γH2AX level was upregulated. Knockdown LIG1 and LIG4 increased DNA damages in trophoblasts, which further induced apoptosis and cell cycle arrest. Serine/arginine-rich splicing factor 1(SRSF1) was reduced in RPL patients and positively correlated with LIG1. Knockdown SRSF1 increased the degradation of LIG1 mRNA which further repressed LIG1 expression. MiR-383 was upregulated in RPL patients and repressed LIG4 expression through interacting with 3'UTR of LIG4 mRNA. The level of miR-383 was found negatively correlated with LIG4 protein level in trophoblasts from RPL patients.

Discussion: LIG1 and LIG4 are downregulated in patients with RPL owing to abnormal RNA degradation and dysregulated miRNA expression. LIG1 and LIG4 downregulation might contribute to the pathophysiological processes of RPL by increasing DNA damages.

Keywords: DNA damage; DNA ligase; Recurrent pregnancy loss; microRNA.