An integrative analysis of an lncRNA-mRNA competing endogenous RNA network to identify functional lncRNAs in uterine leiomyomas with RNA sequencing

Abstract

Objective: To explore the functions of mRNAs and lncRNAs in the occurrence of uterine leiomyomas (ULs) and further clarify the pathogenesis of UL by detecting the differential expression of mRNAs and lncRNAs in 10 cases of UL tissues and surrounding normal myometrial tissues by high-throughput RNA sequencing. Methods: The tissue samples of 10 patients who underwent hysterectomy for UL in Lianyungang Maternal and Child Health Hospital from January 2016 to December 2021 were collected. The differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) were identified and further analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The protein-protein interaction network (PPI) was constructed in Cytoscape software. Functional annotation of the nearby target cis-DEmRNAs of DElncRNAs was performed with the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). Meanwhile, the co-expression network of DElncRNA-DEmRNA was constructed in Cytoscape software. Results: A total of 553 DElncRNAs (283 upregulated DElncRNAs and 270 downregulated DElncRNAs) and 3,293 DEmRNAs (1,632 upregulated DEmRNAs and 1,661 downregulated DEmRNAs) were obtained. GO pathway enrichment analysis revealed that several important pathways were significantly enriched in UL such as blood vessel development, regulation of ion transport, and external encapsulating structure organization. In addition, cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and complement and coagulation cascades were significantly enriched in KEGG pathway enrichment analysis. A total of 409 DElncRNAs-nearby-targeted DEmRNA pairs were detected, which included 118 DElncRNAs and 136 DEmRNAs. Finally, we found that the top two DElncRNAs with the most nearby DEmRNAs were BISPR and AC012531.1. Conclusion: These results suggested that 3,293 DEmRNAs and 553 DElncRNAs were differentially expressed in UL tissue and normal myometrium tissue, which might be candidate-identified therapeutic and prognostic targets for UL and be considered as offering several possible mechanisms and pathogenesis of UL in the future.

Keywords: high-throughput sequencing; long non-coding RNA; mRNAs; miRNAs; uterine leiomyomas.

FIGURE 1

DElncRNA and DEmRNA expression patterns in uterine leiomyoma tissues are related to those in surrounding normal myometrial tissues. (A) Hierarchical cluster analysis of all differentially expressed DElncRNAs. (B) Hierarchical cluster analysis of all differentially expressed DEmRNAs; each column represents a sample, and each row represents a lncRNA and mRNA. The color scale indicates relative expression: upregulation (red) and downregulation (blue). A represents the uterine leiomyoma tissue group, and B represents the surrounding normal myometrial tissue group. (C) Volcano and scatter plots demonstrate differential expression of DElncRNAs between two different conditions. (D) Volcano and scatter plots demonstrate differential expression of DEmRNAs between two different conditions; red points indicate upregulated expression, while green points indicate downregulated expression. The values plotted on the X and Y axes are the averaged normalized signal values of each group (log2 scaled). FC ≥ 2 and p ≤ 0.05 were regarded as the differentially expressed DElncRNAs and DEmRNAs.

FIGURE 2

Functional annotation analysis for the validated DEmRNA gathering genes. (A) GO analysis. (B) KEGG pathway analysis. The vertical axis shows the annotated functions of the target genes. The horizontal axes show the enrichment score (−log10 transformed p value) and the gene number of each cluster, respectively. Only the top 10 significantly enriched clusters are included.

FIGURE 3

Protein–protein interaction (PPI) network analysis. The network comprising the top upregulated (red triangles) and downregulated lncRNAs (green inverted arrows) and their target mRNAs is presented.

FIGURE 4

Cis-nearby-targeted DEmRNAs of DElncRNAs. There were 409 DElncRNA–nearby-targeted DEmRNA pairs, including 118 DElncRNAs and 136 DEmRNAs.

FIGURE 5

Functional annotation analysis for the validated cis-nearby-targeted DEmRNAs of DElncRNA gathering genes. (A) GO analysis. (B) KEGG pathway analysis. The vertical axis shows the annotated functions of the target genes. The horizontal axes show the enrichment score (−log10 transformed p value) and the gene number of each cluster, respectively. Only the top 10 significantly enriched clusters are included.