Identification of circRNA Expression Profile and Potential Systemic Immune Imbalance Modulation in Premature Rupture of Membranes

Abstract

Premature rupture of membrane (PROM) refers to the rupture of membranes before the onset of labor which increases the risk of perinatal morbidity and mortality. Recently, circular RNAs (circRNAs) have emerged as promising regulators of diverse diseases. However, the circRNA expression profiles and potential circRNA-miRNA-mRNA regulatory mechanisms in PROM remain enigmatic. In this study, we displayed the expression profiles of circRNAs and mRNAs in plasma and fetal membranes of PROM and normal control (NC) groups based on circRNA microarray, the Gene Expression Omnibus database, and NCBI's Sequence Read Archive. A total of 1,459 differentially expressed circRNAs (DECs) in PROM were identified, with 406 upregulated and 1,053 downregulated. Then, we constructed the circRNA-miRNA-mRNA network in PROM, encompassing 22 circRNA-miRNA pairs and 128 miRNA-mRNA pairs. Based on the analysis of gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene set enrichment analysis (GSEA), DECs were implicated in immune-related pathways, with certain alterations persisting even postpartum. Notably, 11 host genes shared by DECs of fetal membrane tissue and prenatal plasma in PROM were significantly implicated in inflammatory processes and extracellular matrix regulation. Our results suggest that structurally stable circRNAs may predispose to PROM by mediating systemic immune imbalances, including peripheral leukocyte disorganization, local immune imbalance at the maternal-fetal interface, and local collagen disruption. This is the first time to decipher a landscape on circRNAs of PROM, reveals the pathogenic cause of PROM from the perspective of circRNA, and opens up a new direction for the diagnosis and treatment of PROM.

Figure 1

The differentially expressed circRNAs between the PROM and NC groups before delivery: (a) the normalized circRNA expression level (log2-transformed signal intensity) in each sample; (b) DEcircRNAs with FC > 2.0 and P-value < 0.05; (c) the expression profiles of DEcircRNAs in PROM and NC groups before delivery; (d) chromosomal distribution pattern of DEcircRNAs.

Figure 2

The top DEcircRNAs and their regulated network: (a) the top 20 circRNAs which are selected by the SVM algorithm (P-value < 0.05); (b) the expression level of the top circRNAs in the PROM and NC groups ( ^∗P-value < 0.05;  ^∗∗P-value < 0.01;  ^∗∗∗P-value < 0.001); (c) the expression profiles of DEmiRNAs between the PROM and NC groups (FC > 2 and P-value < 0.05); (d) the expression profiles of DEmRNAs between the PROM and NC groups (FC > 1.5 and P-value < 0.05); (e) the intersection of target miRNAs and DEmiRNAs; (f) the intersection of target mRNAs and DEmRNAs; (g) the ceRNA (circRNA–miRNA–mRNA) regulatory network of top circRNAs in PROM (red squares: upregulated circRNAs; blue squares: downregulated circRNAs; yellow hexagons: miRNAs; dark blue circles: mRNAs).

Figure 3

The top circRNAs-related biological processes: (a) the enriched KEGG terms of genes which are regulated by the top circRNAs; (b) the enriched GO terms of genes which are regulated by the top circRNAs. The linear regression analysis for the immune cells and upregulated (c) and downregulated (d) circRNAs.

Figure 4

The differentially expressed circRNAs between the PROM and NC groups after delivery: (a) DEcircRNAs with FC > 2.0 and P-value < 0.05; (b) the intersection of DEcircRNAs of prenatal and postnatal samples; (c) the expression profiles of intersection DEcircRNAs; (d) the intersection circRNAs-related biological processes; (e) the log2FC of the top circRNAs in prenatal and postnatal samples; (f) the linear regression analysis for the immune cells and has_circ-0096021.

Figure 5

The differentially expressed circRNAs in the fetal membrane between the PROM and NC groups: (a) DEcircRNAs with FC > 2.0 and P-value < 0.05; (b) the intersection of DEcircRNAs of circulation and fetal membrane; (c) the log2FC of the intersection DEcircRNA and mRNAs derived from their host genes; (d) the intersection circRNAs-related biological processes; (e) the significantly dysregulated biological processes in the fetal membrane of PROM; (f) the correlations between the intersection circRNAs and the significantly dysregulated biological processes in the fetal membrane of PROM (the colors indicate the correlation coefficients).