Construction of implantation failure related lncRNA-mRNA network and identification of lncRNA biomarkers for predicting endometrial receptivity

Abstract

Insufficient endometrial receptivity is a major factor leading to implantation failure (IF), and the traditional way of morphological observation of endometrium cannot determine the condition of receptivity sufficiently. Considering that long-noncoding RNAs (lncRNAs) regulate endometrial receptivity and competing endogenous RNA (ceRNA) mechanism works in plenty of biological processes, ceRNA is likely to function in the pathology of IF. In the present study, we aim to construct an implantation failure related lncRNA-mRNA network (IFLMN), and to identify the key lncRNAs as the candidates for predicting endometrial receptivity. The global background network was constructed based on the presumed lncRNA-miRNA and miRNA-mRNA pairs obtained from lncRNASNP and miRTarBase. Differentially expressed genes (DEGs) of IF were calculated using the data of GSE26787, and then re-annotated as differentially expressed mRNAs (DEMs) and lncRNAs (DELs). IFLMN was constructed by hypergeometric test, including 255 lncRNA-mRNA pairs, 10 lncRNAs, and 212 mRNAs. Topological analysis determined the key lncRNAs with the highest centroid. Functional enrichment analyses were performed by unsupervised clustering, GO classification, KEGG pathway, and co-expression module analyses, achieving six key lncRNAs and their ceRNA sub-networks, which were involved in immunological activity, growth factor binding, vascular proliferation, apoptosis, and steroid biosynthesis in uterus and prepared endometrium for embryo implantation. Sixteen endometrial samples were collected during mid-luteal phase, including 8 recurrent implantation failure (RIF) or recurrent miscarriage (RM) women and 8 controls who conceived successfully. Quantitative real-time PCR was performed to compare the expression of the above six lncRNAs, which validated that the expression of all these lncRNAs was significantly elevated in endometrium of RIF/RM patients. Further studies are needed to investigate the underlying mechanism, and the lncRNAs may be developed into predictive biomarkers for endometrial receptivity.

Keywords: Implantation failure; biomarkers; competing endogenous RNA (ceRNA); endometrial receptivity; long non-coding RNA (lncRNA); messenger RNA (mRNA); microRNA (miRNA).

Figure 1

Workflow of the present study. First, the lncRNA-miRNA interact pairs were obtained from lncRNASNP, and the miRNA-mRNA interact pairs were obtained from miRTarBase. The pairs shared the same miRNA were combined to make up lncRNA-miRNA-mRNA triple pairs and the global triple network. Second, gene expression data were downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified by Bioconductor limma package with fold change > 2 and adjusted P < 0.05. The DEGs were re-annotated and identified as differentially expressed mRNAs (DEMs) and lncRNAs (DELs). Third, IFLMN were extracted from the global network by hypergeometric test with P < 0.01. Fourth, the functional enrichment was mined by unsupervised clustering, GO, and KEGG annotation. Fifth, the topological features of IFLMN were analyzed and four key lncRNAs were selected. Sixth, construction of the sub-network with key lncRNAs and enrichment analysis. Seventh, extracting the co-expression modules with bi-directional clustering and enrichment analysis. Eighth, validation of the expression of key lncRNAs in endometrium with real-time PCR.

Figure 2

Implantation failure related lncRNA-mRNA network (IFLMN) and enrichment analysis. A. The blue concave quadrilateral represents lncRNA and the green diamond represents mRNA. Grey edges indicate lncRNA-mRNA interactions. Size of the node represents the degrees of the nodes in IFLMN. The lncRNAs marked with purple labels are the four most central lncRNAs. B. GO analysis of IFLMN. C. KEGG pathway analysis of IFLMN. The dotted lines emphasize the annotations that have been proved to work in endometrial receptivity.

Figure 3

Custer analysis of implantation failure related lncRNA-mRNA network (IFLMN) and its co-expression network. A. The clustering of the IFLMN genes. Unsupervised clustering of IFLMN genes could discriminate between fertile and IF groups. B. The Venn of predicted transcription factors (TFs) of the four co-expression lncRNAs. There are 25 common TFs and TFs involved in the sexual hormone functions were marked in brown. C. GO enrichment analysis of co-expression mRNAs. D. KEGG pathway analysis of co-expression mRNAs. The dotted lines emphasize the annotations that have been proved to work in endometrial receptivity.

Figure 4

Topological characteristics of implantation failure related lncRNA-mRNA network (IFLMN) and ceRNA sub-network of NONHSAT212577.1. A. Topological characteristics of IFLMN. There are four genes existed simultaneously in top 10 lists of degree, betweenness, and closeness. B. Distribution of degree. The top four genes with highest degree were four lncRNAs. C. Molecular function enrichment of NONHSAT212577.1. D. Biological process enrichment of NONHSAT212577.1. E. KEGG enrichment of NONHSAT212577.1. F. The co-expression subnet of NONHSAT212577.1. The dotted lines emphasize the co-expressed genes. G. The ceRNA network of NONHSAT212577.1. The dotted lines emphasize the annotations that have been proved to work in endometrial receptivity.

Figure 5

The ceRNA sub-network of NONHSAT083203.2. A. Biological process enrichment of NONHSAT083203.2. B. KEGG enrichment of NONHSAT083203.2. C. The co-expression subnet of NONHSAT083203.2. The dotted lines emphasize the co-expressed genes. D. The ceRNA network of NONHSAT083203.2.

Figure 6

The ceRNA sub-network of NONHSAT193031.1. A. Biological process enrichment of NONHSAT193031.1. B. KEGG enrichment of NONHSAT193031.1. C. The co-expression subnet of NONHSAT193031.1. The dotted lines emphasize the co-expressed genes. D. The ceRNA network of NONHSAT193031.1.

Figure 7

The ceRNA sub-network of NONHSAT035952.2. A. Biological process enrichment of NONHSAT035952.2. B. KEGG enrichment of NONHSAT035952.2. C. The co-expression subnet of NONHSAT035952.2. The dotted lines emphasize the co-expressed genes. D. The ceRNA network of NONHSAT035952.2.

Figure 8

The co-expression networks in implantation failure related lncRNA-mRNA network (IFLMN). A. The bidirectional clustering of IFLMN. The genes with green bar were included in Module 1 and the genes with blue bar were included in Module 2. B. Module 1 of ceRNA networks. Module 1 included 3 lncRNAs and 29 mRNAs. C. Module 2 of ceRNA networks. Module 2 included 2 lncRNAs and 13 mRNAs. D. GO classification of Module 1. E. KEGG pathway of Module 1. F. GO classification of Module 2. G. KEGG pathway of Module 2.

Figure 9

Expression of lncRNAs in endometrium. The six key lncRNAs were assayed by real-time PCR on a sample set of 8 RIF patients and 8 controls. The expression levels of lncRNAs in RIF increased significantly compared to fertile controls. * P < 0.05.