Downregulation of miR-1388 Regulates the Expression of Antiviral Genes via Tumor Necrosis Factor Receptor (TNFR)-Associated Factor 3 Targeting Following poly(I:C) Stimulation in Silver Carp (Hypophthalmichthys molitrix)

Abstract

MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal physiological functions in fish. Nevertheless, the specific physiological role of miRNAs in lower vertebrates, particularly in comparison to mammals, remains elusive. Additionally, the mechanisms underlying the control of antiviral responses triggered by viral stimulation in fish are still not fully understood. In this study, we investigated the regulatory impact of miR-1388 on the signaling pathway mediated by IFN regulatory factor 3 (IRF3). Our findings revealed that following stimulation with the viral analog poly(I:C), the expression of miR-1388 was significantly upregulated in primary immune tissues and macrophages. Through a dual luciferase reporter assay, we corroborated a direct targeting relationship between miR-1388 and tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Furthermore, our study demonstrated a distinct negative post-transcriptional correlation between miR-1388 and TRAF3. We observed a significant negative post-transcriptional regulatory association between miR-1388 and the levels of antiviral genes following poly(I:C) stimulation. Utilizing reporter plasmids, we elucidated the role of miR-1388 in the antiviral signaling pathway activated by TRAF3. By intervening with siRNA-TRAF3, we validated that miR-1388 regulates the expression of antiviral genes and the production of type I interferons (IFN-Is) through its interaction with TRAF3. Collectively, our experiments highlight the regulatory influence of miR-1388 on the IRF3-mediated signaling pathway by targeting TRAF3 post poly(I:C) stimulation. These findings provide compelling evidence for enhancing our understanding of the mechanisms through which fish miRNAs participate in immune responses.

Keywords: TRAF3; innate immunity; miR-1388; poly(I:C); silver carp.

Figure 1

Poly(I:C) stimulation induces the downregulation of miR-1388 expression. (A,B) The mRNA expression levels of antiviral genes in the kidney tissues of silver carp treated with poly(I:C) were measured using qRT-PCR. (C) The expression level of miR-1388 in various tissues of silver carp stimulated by poly(I:C) was determined by qRT-PCR. (D) The expression level of miR-1388 in macrophages treated with different concentrations of poly(I:C) was assessed by qRT-PCR. (E) The expression level of miR-1388 in macrophages following treatment with poly(I:C) for varying durations was examined using qRT-PCR. Each experiment was replicated at least three times, and the results are presented as the means ± SE. **, p < 0.01 versus the control group.

Figure 2

miR-1388’s impact on the antiviral genes stimulated by poly(I:C). (A) Following a 48 h transfection of miR-1388 mimics, the expression level of miR-1388 in macrophages was quantified using qRT-PCR. (B) The expression levels of antiviral genes in macrophages were assessed by qRT-PCR after a 48 h transfection with mimics. (C) Additionally, macrophages were transfected with mimics for 48 h and then stimulated with poly(I:C) for 6 h to examine the impact on antiviral gene expression, which was measured using qRT-PCR. (D) The expression level of miR-1388 in macrophages was determined by qRT-PCR following the 48 h transfection of the miR-1388 inhibitor. (E) The effect of the inhibitor on antiviral gene expression was evaluated by measuring their levels in macrophages using qRT-PCR after a 48 h transfection. (F) Moreover, macrophages transfected with the inhibitor for 48 h were stimulated with poly(I:C) for 6 h to assess its influence on antiviral gene expression, which was quantified by qRT-PCR. All experiments were performed at least three times, and the results are presented as the means ± SE. Statistical analysis revealed significant differences compared to the control group: *, p < 0.05; **, p < 0.01.

Figure 3

Analysis of miR-1388 targeting TRAF3 3′UTR. (A) Utilization of bioinformatics tools to predict potential binding sites of miR-1388 in the 3′UTR of TRAF3. (B) Co-transfection of wild-type or mutant TRAF3 3′UTR dual-luciferase reporter plasmids with miR-1388 or NC into HEK293T cells. Subsequently, luciferase activity was assessed after 48 h. (C) Co-transfection of wild-type TRAF3 3′UTR dual-luciferase reporter plasmids with varying concentrations of miR-1388 (0, 25, 50, 100 nM) or NC (100, 50, 25, 0 nM) into HEK293T cells. After 48 h, luciferase activity was measured. (D) Co-transfection of wild-type TRAF3 3′UTR dual-luciferase reporter plasmids with miR-1388, miR-1388-i, NC, NC-i into HEK293T cells. Following 48 h incubation, luciferase activity was quantified. (E) Co-transfection of wild-type or mutant mVenus-TRAF3-3′UTR constructs with miR-1388 or NC into HEK293T cells. Post 48 h, fluorescence intensity was evaluated using an enzyme-linked instrument. The scale bar is set at 100 μm, and the original magnification is ×10. All luciferase activities were normalized to the Renilla luciferase activity. Each experiment was conducted at least thrice, and the results are presented as the means ± SE. *, p < 0.05; **, p < 0.01 compared to the control group.

Figure 4

Expression levels and functions of TRAF3 in innate immune responses. (A) The mRNA expression levels of TRAF3 in various tissues of silver carp were detected using RT-qPCR after 6 h of poly(I:C) stimulation. (B) The mRNA expression levels of TRAF3 in macrophages were detected using RT-qPCR after stimulation with different concentrations of poly(I:C). (C) The protein expression levels of TRAF3 in macrophages were detected using Western blotting after stimulation with different concentrations of poly(I:C). (D) The mRNA expression levels of TRAF3 in macrophages were detected using RT-qPCR at different time points (0, 6, 12, 24 h) after poly(I:C) stimulation. (E) Luciferase activity was measured after the co-transfection of IRF3, ISRE luciferase reporter plasmid with TRAF3 overexpression plasmid or EV in HEK293T cells for 48 h. All luciferase activities were normalized to the Renilla luciferase activity. Each experiment was conducted at least three times, and the results are presented as the means ± SE. **, p < 0.01 compared to the control group. Original images can be found in Figure S1.

Figure 5

Expression analysis of the miR-1388 and TRAF3 interaction. (A) The mRNA expression levels of TRAF3 in HEK293T cells were determined using RT-qPCR after co-transfection with different concentrations of miR-1388 or NC for 48 h. (B) The protein expression levels of TRAF3 in HEK293T cells were assessed following co-transfection with different concentrations of miR-1388 or NC for 48 h. (C) The mRNA expression levels of TRAF3- in macrophages were measured using RT-qPCR after transfection with miR-1388 or NC for 48 h. (D) The protein expression levels of TRAF3 in macrophages were evaluated after transfection with different concentrations of miR-1388 or NC for 48 h. (E) The mRNA expression levels of TRAF3- in macrophages were quantified using RT-qPCR after transfection with miR-1388-i or NC-i for 48 h. (F) The protein expression levels of TRAF3 in macrophages were analyzed following transfection with different concentrations of miR-1388-i or NC-i for 48 h. Each experiment was performed at least three times, and the results are presented as the means ± SE. **, p < 0.01 compared to the control group. Original images can be found in Figure S1.

Figure 6

Regulation of antiviral genes by miR-1388 via TRAF3 targeting. (A) Luciferase activity of IRF3 and ISRE luciferase reporter plasmids co-transfected with TRAF3 overexpression plasmid, miR-1388, or NC was measured in HEK293T cells after 48 h. (B) Luciferase activity of IRF3 and ISRE luciferase reporter plasmids co-transfected with TRAF3 overexpression plasmid, miR-1388, or NC was measured in HEK293T cells after 48 h, followed by stimulation with poly(I:C) for 6 h. (C) Assessment of TRAF3 protein expression levels in macrophages after transfection with an increasing amount of si-TRAF3 or NC for 48 h. (D) mRNA expression levels of antiviral genes in macrophages were determined using RT-qPCR after transfection with si-TRAF3or NC for 48 h, followed by stimulation with poly(I:C) for 6 h. All luciferase activities were normalized to Renilla luciferase activity. Each experiment was performed at least three times, and results are presented as means ± SE. *, p < 0.05; **, p < 0.01 compared to the control group. Original images can be found in Figure S1.