The Circ-CYP24A1-miR-224-PRLR Axis Impairs Cell Proliferation and Apoptosis in Recurrent Miscarriage.

Yan Su,Rufei Gao,Taihang Liu,Hongbo Qi,Xueqing Liu,Yingxiong Wang,Yubin Ding,Chunli Li,Xuemei Chen,Cong Li,Junlin He,Xiaoli Liu,Jiani Xu

doi:10.3389/fphys.2022.778116

Abstract

AimRecurrent miscarriage (RM) is associated with numerous clinical factors. However, some RM occurred without specific factors. It has been revealed that some molecules such as hormones, miRNAs, and transcription factors are involved in RM by regulating proliferation, apoptosis, etc. However, the mechanism of RM has yet to be identified clearly. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that often act as sponges for miRNAs or binds to proteins involved in biological processes. However, the functional role of circRNAs in the uterine decidua of patients with early RM is still unclear. In this study, we aimed to investigate the mechanisms of circ-CYP24A1 in RM.MethodsThe Dual-Luciferase Activity Assay was designed to analyze the bonding between circ-CYP24A1 and miR-224, and miR-224 and prolactin receptor (PRLR) mRNA 3′UTR. In situ hybridization (ISH) and immunohistochemistry (IHC) were used to observe the expression of circ-CYP24A1 and PRLR in the decidua. Rescue experiments were performed to investigate the regulating effects of circ-CYP24A1, miR-224, and PRLR. Western blotting was conducted to test the expression level of PRLR. The proliferation and apoptosis-related markers in Ishikawa cells were analyzed using CCK8, immunofluorescence staining, and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay.ResultsIn this study, based on the microarray analysis data, we identified a high level of circ-CYP24A1 and PRLR in the decidua of patients with early RM. Based on the bioinformatics prediction, the binding relationship between circ-CYP24A1 and miR-224, as well as miR-224 and PRLR, were verified. Functional experiments demonstrated that circ-CYP24A1 regulated proliferation and apoptosis by binding to and inhibiting miR-224, resulting in increased PRLR expression. Taken together, this study provides new insights into the mechanism of RM.ConclusionIn this study, we found that circ-CYP24A1 plays a role in RM by impairing the balance of cell proliferation and apoptosis by sponging miR-224, thereby regulating PRLR.

Highlights

Recurrent miscarriage (RM) is defined as three or more consecutive miscarriages within 24 weeks
In our previous study, using bioinformatics predictions from miRanda and the TargetScan database, we found that circ-CYP24A1 may act as a sponge for miR-224 and regulate the expression of the prolactin receptor (PRLR) gene
We used ISH to verify the expression of circ-CYP24A1 in the decidua of patients with early RM and unintended pregnant women

Summary

Introduction

Recurrent miscarriage (RM) is defined as three or more consecutive miscarriages within 24 weeks. Miscarriage occurring before the 12th week of pregnancy is defined as early RM. Decidualization is driven by timely proliferation, apoptosis, and differentiation of endometrial stromal cells and is marked by high expression of prolactin (PRL) and insulin growth factor binding protein-1 (IGFBP1) (Fabi et al, 2017; Ochoa-Bernal and Fazleabas, 2020). RM has been associated with abnormal proliferation, apoptosis, and differentiation of endometrial stromal cells (Diniz-da-Costa et al, 2021; Meng et al, 2021). Hong et al (2018) showed that the microRNA miR-378a-3p plays a role in RM by regulating the proliferation and apoptosis of decidual cells. It has been shown that hormones, miRNAs, and transcription factors participate in RM by regulating proliferation and apoptosis (Meng et al, 2021), the mechanisms underlying RM have yet to be identified clearly. The upstream molecular mechanism of miRNA in RM is still unclear

Objectives

Methods

Results

Conclusion