Differential expression profiling of spleen microRNAs in response to two distinct type II interferons in Tetraodon nigroviridis

Abstract

MicroRNAs are endogenous, small non-coding RNAs approximately 18-26 nucleotides in length that regulate target gene expression at the post-transcription level. Interferon-γ (IFN-γ) is a Th1 cytokine that is involved in both the innate and adaptive immune responses. We previously identified two IFN-γ genes in green-spotted puffer fish (Tetraodon nigroviridis). To determine whether miRNAs participate in IFN-γ-related immune responses, T. nigroviridis spleen cells were treated with recombinant IFN-γ isoforms, and a Solexa high-throughput sequencing method was used to identify miRNAs. In total, 1,556, 1,538 and 1,573 miRNAs were found in the three samples, and differentially expressed miRNAs were determined. In total, 398 miRNAs were differentially expressed after rIFN-γ1 treatment, and 438 miRNAs were differentially expressed after rIFN-γ2 treatment; additionally, 403 miRNAs were differentially expressed between the treatment groups. Ten differentially expressed miRNAs were chosen for validation using qRT-PCR. Target genes for the differentially expressed miRNAs were predicted, and GO and KEGG analyses were performed. This study provides basic knowledge regarding fish IFN-γ-induced miRNAs and offers clues for further studies into the mechanisms underlying fish IFN-γ-mediated immune responses.

Figure 1. Read length distribution of the clean reads from the (A) Sp-con, (B) Sp-γ1, and (C) SP-γ2 groups.

Figure 2. Small RNA tags matched to the genome by SOAP.

The figure shows the expression and genomic distribution of miRNAs identified in the (A) Sp-con, (B) Sp-γ1, (C) SP-γ2 groups.

Figure 3. Expression of the identified miRNAs in the three different samples using hierarchical clustering.

Highly expressed miRNAs are indicated in red, and miRNAs with low expression are indicated in green; grey indicates missing data. The absolute signal intensity ranged from −4.0 to +4.0.

Figure 4. Log2-ratio scatter plot of the differentially expressed miRNAs between (A) the Sp-con and Sp-γ1 groups, (B) the Sp-con and Sp-γ2 groups, and (C) the Sp-γ1 and SP-γ2 groups.

Each point in the figure represents a miRNA. The X and Y axes show the expression levels in the two samples. The red points represent miRNAs with a ratio >2, the blue points represent miRNAs with a ratio >1/2 and ≤2, and the green points represent miRNAs with a ratio ≤1/2.

Figure 5. Normalized read counts for selected miRNAs.

(A): Normalized read counts for miR-124b-3p, miR-142-3p, miR-145b, miR-17, miR-29d-3p, and miR-346 following IFNγ1 treatment, compared to the control group. (B): Normalized read counts of miR-106a-3p, miR-124b-3p, miR-132-3p, miR-17, miR-223-5p, and miR-378 following IFNγ2 treatment, compared to the control group.

Figure 6. Quantitative real-time PCR validation of selected miRNAs.

(A): expression of miR-124b-3p, miR-142-3p, miR-145b, miR-17, miR-29d-3p, and miR-346 following IFNγ1 treatment, compared to the control group. (B): expression of miR-106a-3p, miR-124b-3p, miR-132-3p, miR-17, miR-223-5p, and miR-378 following IFNγ2 treatment, compared to the control group. The amount of each miRNA was normalized to that of U6 snRNA and is presented as the relative fold change (n = 3, mean ± SEM). Significant differences between the control and treatment groups are indicated (*P<0.05; **P<0.01; ***P<0.001).

Figure 7. KOG classes of predicted targets for Tetraodon nigroviridis miRNAs.