RNA sequencing analysis of altered expression of long noncoding RNAs associated with Schistosoma japonicum infection in the murine liver and spleen

Abstract

Background: Schistosomiasis is a chronic, debilitating infectious disease caused by members of the genus Schistosoma. Previous findings have suggested a relationship between infection with Schistosoma spp. and alterations in the liver and spleen of infected animals. Recent reports have shown the regulatory role of noncoding RNAs, such as long noncoding RNAs (lncRNAs), in different biological processes. However, little is known about the role of lncRNAs in the mouse liver and spleen during Schistosoma japonicum infection.

Methods: In this study, we identified and investigated lncRNAs using standard RNA sequencing (RNA-Seq). The biological functions of the altered expression of lncRNAs and their target genes were predicted using bioinformatics. Ten dysregulated lncRNAs were selected randomly and validated in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) experiments.

Results: Our study identified 29,845 and 33,788 lncRNAs from the liver and spleen, respectively, of which 212 were novel lncRNAs. We observed that 759 and 789 of the lncRNAs were differentially expressed in the respective organs. The RT-qPCR results correlated well with the sequencing data. In the liver, 657 differentially expressed lncRNAs were predicted to target 2548 protein-coding genes, whereas in the spleen 660 differentially expressed lncRNAs were predicted to target 2673 protein-coding genes. Moreover, functional annotation showed that the target genes of the differentially expressed lncRNAs were associated with cellular processes, metabolic processes, and binding, and were significantly enriched in metabolic pathways, the cell cycle, ubiquitin-mediated proteolysis, and pathways in cancer.

Conclusions: Our study showed that numerous lncRNAs were differentially expressed in S. japonicum-infected liver and spleen compared to control liver and spleen; this suggested that lncRNAs may be involved in pathogenesis in the liver and spleen during S. japonicum infection.

Keywords: Liver; Long noncoding RNA; Pathogenesis; RNA sequencing; Schistosoma japonicum; Spleen.

Fig. 1a–d

Classification and characteristics of long noncoding RNAs (lncRNAs) identified in infected and control murine livers and spleens. a Annotations of lncRNAs identified in infected and control liver and spleen specimens. b Number of exons per transcript for mRNAs and lncRNAs in livers and spleens of mice. c Box plot showing the transcript size distributions for mRNAs and lncRNAs in livers and spleens of mice. d Violin plot for lncRNA and mRNAs expression in livers and spleens of mice. TPM Transcripts per million

Fig. 2a–d

Differentially expressed mRNAs and lncRNAs in infected murine livers and spleens. a Volcano plot of differentially expressed mRNAs in livers of infected versus control mice. b Volcano plot of differentially expressed mRNAs in spleens of infected versus control mice. c Volcano plot of differentially expressed lncRNAs in livers of infected versus control mice. d Volcano plot of differentially expressed lncRNAs in spleens of infected versus control mice. The x-axis represents the log2 (fold change) values of the differentially expressed mRNAs/lncRNAs, and the y-axis represents the −log10 (q-value) values of the differentially expressed mRNAs/lncRNAs

Fig. 3

Enrichment of Gene Ontology terms for dysregulated lncRNA target genes in infected liver (a) and spleen (b) versus the corresponding control. The horizontal axis represents the annotation terms of interest, and the vertical axis shows the number of genes annotated to each term

Fig. 4a, b

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed lncRNA targets in livers and spleens of infected mice. The top 30 KEGG terms associated with the differentially expressed lncRNA targets in liver (a) and spleen (b) of S. japonicum-infected mice versus control mice. The vertical axis shows the pathway names, and the horizontal axis shows the rich factors of KEGG pathway enrichment. The rich factor was calculated as (the number of differentially expressed genes associated with a specific pathway term/the number of all differentially expressed genes in the pathway database)/(the number of genes associated with a specific pathway term/the total number of genes in the pathway database). The size of each dot indicates the number of differentially expressed genes in the pathway, and the color corresponds to the different q-value range

Fig. 5a, b

Co-expression network of differentially expressed lncRNAs and correlated mRNAs. a The 50 most differentially expressed lncRNAs and 107 dysregulated, correlated mRNAs in the livers of mice infected with S. japonicum. b The 50 most significantly expressed lncRNAs and 68 dysregulated, correlated mRNAs in the spleens of mice infected with S. japonicum. The circles represent lncRNAs and the diamonds represent target mRNAs. The sliding color scale from green to red indicates low to high expression, respectively. The sizes of the circles and diamonds indicate the number of associated genes

Fig. 6a–d

Verification of differentially expressed lncRNAs and their putative targets in livers and spleens of mice infected with S. japonicum by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Relative expression levels of lncRNAs in livers (a) and spleens (b) of mice at 25 days post-infection. Relative expression levels of the putative targets for selected lncRNAs in livers (c) and spleens (d) of mice at 25 days post-infection. The relative expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. XCR1 Chemokine C motif receptor 1, VCAM1 vascular cell adhesion molecule 1, SAMHD1 SAM domain and HD domain 1, CCR5 chemokine C–C motif receptor 5, GLCC1 glucocorticoid-induced transcript 1, IRS1 insulin receptor substrate 1, EXO1 exonuclease 1, FRY furry homolog Drosophila, CCDC137, coiled coil domain containing 137, TTYH1 tweety homolog 1 Drosophila. The results are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01 *** p <   0.01, *** p < 0.001, **** p < 0.0001, NC no statistically significant change

Fig. 7a, b

Differentially expressed lncRNAs and related putative targets in livers of mice infected with S. japonicum examined by RT-qPCR. a Relative expression levels of selected lncRNAs in the livers of mice infected with S. japonicum at 64 days post-infection. b Relative expression levels of several putative targets for lncRNAs in the livers of mice infected with S. japonicum at 64 days post-infection. The relative expression levels were normalized to GAPDH gene expression. The results are expressed as the mean ± SEM. **p < 0.01 ***p < 0.001, ****p < 0.0001. For abbreviations, see Figs. 1 and 6