Integrating miRNA and mRNA Profiling to Assess the Potential miRNA-mRNA Modules Linked With Testicular Immune Homeostasis in Sheep

Abstract

Beyond its well-known role in spermatogenesis and androgen production, mammalian testes are increasingly recognized as an immune-privileged organ for protecting autoantigenic germ cells, especially meiotic and postmeiotic germ cells, from systemic immune responses. Despite its importance, the molecular mechanisms underlying this regulation in mammals, including sheep, are far from known. In this study, we searched for the genes associated with testicular immune privilege and assessed their possible modulating mechanisms by analyzing systematic profiling of mRNAs and miRNAs on testicular tissues derived from prepubertal and postpubertal Tibetan sheep acquired by RNA sequencing. We identified 1,118 differentially expressed (DE) mRNAs associated with immunity (245 increased mRNAs and 873 decreased mRNAs) and 715 DE miRNAs (561 increased miRNAs and 154 decreased miRNAs) in postpubertal testes compared with prepuberty. qPCR validations for 20 DE mRNAs and 16 miRNAs showed that the RNA-seq results are reliable. By using Western blot, the postpubertal testes exhibited decreased protein abundance of CD19 and TGFBR2 (two proteins encoded by DE mRNAs) when compared with prepuberty, consistent with mRNA levels. The subsequent immunofluorescent staining showed that the positive signals for the CD19 protein were observed mainly in Sertoli cells and the basement membrane of pre- and postpubertal testes, as well as the prepubertal testicular vascular endothelium. The TGFBR2 protein was found mostly in interstitial cells and germ cells of pre- and postpubertal testes. Functional enrichment analysis indicated that DE mRNAs were mainly enriched in biological processes or pathways strongly associated with the blood-testis barrier (BTB) function. Many decreased mRNAs with low expression abundance were significantly enriched in pathways related to immune response. Also, multiple key miRNA-target negative correlation regulatory networks were subsequently established. Furthermore, we verified the target associations between either oar-miR-29b or oar-miR-1185-3p and ITGB1 by dual-luciferase reporter assay. Finally, a putative schematic model of the miRNA-mRNA-pathway network mediated by immune homeostasis-related genes was proposed to show their potential regulatory roles in sheep testicular privilege. Taken together, we conclude that many immune-related genes identified in this study are negatively regulated by potential miRNAs to participate in the homeostatic regulation of testicular immune privilege of sheep by sustaining BTB function and inhibiting immune responses under normal physiological conditions. This work offers the first global view of the expression profiles of miRNAs/mRNAs involved in sheep testicular immune privilege and how the genes potentially contribute to immune-homeostatic maintenance.

Keywords: RNA-seq; Tibetan sheep; blood-testis barrier; immune privilege; miRNA; testis.

Figure 1

Differential gene expression analysis between T3M and T1Y. (A) Hierarchical clustering dendrograms of the biological duplicates. (B) Sample correlation heatmap. (C) Biological processes mediated by DE mRNAs based on Gene Ontology (GO) annotation results. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for DE mRNAs. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep; DE, differentially expressed.

Figure 2

Filtering of DE mRNAs related to immunity. (A) The histogram shows the numbers of the immune-related DE mRNAs. Red and blue represent increased and decreased mRNA abundance in T1Y, respectively. (B) Venn diagram of the immune-related DE mRNAs in two age groups. (C) The clustering heatmap for the immune-related DE mRNAs. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep; DE, differentially expressed.

Figure 3

Functional annotation and enrichment analysis for immune-related DE mRNAs. (A) GO annotation results. (B) Top 20 KEGG pathways enriched by immune-related mRNAs. Y-axis denotes the enrichment factor, which was calculated by dividing the number of enriched genes (mRNAs) in the KEGG pathway by the number of annotated genes in this pathway. (C) Venn diagram of DE mRNAs related to extracellular matrix remodeling, cell junction, cell adhesion, and immune response.

Figure 4

Analysis of DE miRNAs. (A) Numbers of DE miRNAs. (B) Volcano plot of DE miRNAs. Red represents increased miRNA abundance, and green represents decreased miRNA abundance in T1Y relative to T3M. (C) Venn diagram representation of the common and group-specific DE miRNAs. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep; DE, differentially expressed.

Figure 5

Integrated miRNA-target negative correlation regulatory networks. (A) Immune-related gene CD19 was potentially regulated by 24 known miRNAs. (B) Three genes related to the blood–testis barrier function, namely, CLDN11, ITGA6, and ITGB1, were potentially regulated by 87 known miRNAs. (C) Four genes that have been reported as testicular immune homeostasis-related genes, including TGFB1, TGFBR2, TGFBR3, and FASLG, were potentially regulated by 117 known miRNAs. (D) Fifty-one DE genes were potentially regulated by miR-146-5p, an miRNA related to immune homeostasis maintenance. The circle nodes represent miRNAs; the triangle nodes suggest target genes. The differentially expressed miRNAs/target genes are highlighted in red and blue, describing higher and lower expression in the T1Y group compared with the T3M group, respectively. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep.

Figure 6

Verification of DE mRNAs and DE miRNAs by qPCR. (A) qPCR analysis for 20 randomly selected mRNAs. Data represent the mean ± SD. (B) qPCR analysis for 16 randomly selected miRNAs. Data represent the mean ± SD. (C) The comparison of mRNA expression in terms of Log₂ (fold change) as assessed by mRNA sequencing and qPCR. (D) The comparison of mRNA expression in terms of the Log₂ (fold change) as assessed by miRNA sequencing and qPCR. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep; DE, differentially expressed.

Figure 7

Expression and localization patterns of CD19 and TGFBR2 proteins. (A) Western blot analysis. (B) Relative protein expression. Data represent the mean ± SD. (C) Immunofluorescence staining. Blue, DAPI; red/CY3, CD19; green/FITC, TGFBR2. Scale bar, 50 μm. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep; DE, differentially expressed. **P < 0.01.

Figure 8

Validation of targeted associations between either miR-29b or miR-1185-3p and ITGB1. (A) Pearson correlation assessment between oar-miR-29b/oar-miR-1185-3p and ITGB1 expression. (B) Quantitative real-time PCR (qPCR) detection for oar-miR-29b, oar-miR-1185-3p, and ITGB1 expression. (C) Schematic presentation of the pmirGLO dual-luciferase miRNA target expression vector used for constructing ITGB1-3′UTR. (D) A dual-luciferase reporter assay verified the interaction between miR-29b/miR-1185-3p and ITGB1. (Upper panel) The binding sites on ITGB1 3′-UTR and the seed region of miR-29b or miR-1185-3p. (Lower panel) Relative luciferase activity was measured by a dual-luciferase reporter assay. Data represent the mean ± SD. T3M, testes from 3-month-old sheep; T1Y, testes from 1-year-old sheep. **P < 0.01.

Figure 9

A proposed putative model showing the involvement of DE genes and miRNAs in testicular immune homeostasis through related pathways.