Essential role of hsa-miR-203a-3p in type I interferons immune homeostasis during influenza and NDV infection

ABSTRACT

MicroRNAs (miRNAs) are small, non-coding RNA molecules that act as essential post-transcriptional regulators in various biological processes. Many studies suggest that miRNAs may modulate the host’s immune response or even viral replication during infection. We have identified hsa-miR-203a-3p as a key regulatory candidate influencing innate immune responses, based on a comprehensive analysis of publicly available transcriptomic datasets involving H7N9, HCV, or DENV2 infection. Pathway enrichment analysis of microRNA-targeted genes reveals that hsa-miR-203a-3p targets several components of type I interferon signalling and JAK-STAT pathway. In this study, we report a novel role of hsa-miR-203a-3p as it is elevated in response to polyinosinic-polycytidylic acid [poly(I:C)] transfection and infection with RNA viruses Newcastle Disease Virus (NDV) and A/PR8/H1N1 influenza virus. We found that hsa-miR-203a-3p promotes the A/PR8/H1N1 virus replication by suppressing the host’s type-I interferons and interferon-stimulated genes. Our investigation demonstrated that overexpression of hsa-miR-203a-3p led to reduced expression of interferon stimulated genes (ISGs). This regulation is likely mediated through the direct binding of hsa-miR-203a-3p to the 3’ UTRs of Janus-activated kinase 1 (JAK1), STAT1 and several IFN-α transcripts. Collectively, these findings highlight the pivotal role of hsa-miR-203a-3p in immune homoeostasis; it regulates type I IFN signalling and downstream antiviral responses, thereby facilitating A/PR8/H1N1 and NDV infection.

Introduction

RNA viruses, such as influenza, MERS, SARS-CoV, and SARS-CoV-2, have been the cause of several epidemics and pandemics in recent years and, therefore, pose a significant threat to global human health. These viruses are first detected by the host’s innate immune system, which comprises a family of extracellular and intracellular sensors called pattern recognition receptors (PRRs) [1,2]. PRRs detects conserved molecular signatures associated with viruses, such as viral proteins, RNA with 5'-triphosphate ends, hypomethylated or unmethylated viral DNA, single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA) [3,4]. Sensing of these molecules by PRRs activates a plethora of downstream signalling pathways, culminating in the activation of transcription factor interferon (IFN) regulatory factor (IRFs) and nuclear factor-kB (NF-kB) for the induction of Type-I interferons and pro-inflammatory cytokines [5,6]. These transcription factors, in turn, lead to the expression of several key coding and non-coding transcripts that collectively contribute to the development of an antiviral state [7]. This complex process of antiviral signalling is regulated at the post-transcriptional and post-translational levels. Post-translational regulation primarily occurs through the ubiquitination or phosphorylation of transcription factors such as IRFs, JAKs, and STATs [8–10]. In contrast, post-transcriptional regulation is largely mediated by various non-coding RNAs, including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) [11–13]. miRNAs are a class of single-stranded non-coding RNAs (ncRNAs) ranging from 18 to 21 nucleotides in length, playing a pivotal role in fine-tuning antiviral immune responses through a multiprotein RNA-induced silencing complex (RISC) [14].

miRNAs regulate gene expression by binding to the 3’UTR (3’ untranslated region) region of target transcripts and repressing their translation. Several transcriptomic studies have shown that the virus infection induces the expression of many miRNAs, targets innate immune signalling pathways, and modulates the antiviral response [15,16]. Notably, several studies have demonstrated that miRNAs can also directly bind to the genomic RNA of RNA viruses, thereby directly modulating viral replication [17–21]. Upon infection, the genetic material of RNA viruses can be sensed by Toll-like receptor 3 (TLR3), TLR7, and TLR8 in the endosomal compartment, or by cytosolic sensors such as RIG-I and MDA5, inducing type I interferons necessary to mount an effective antiviral immune response [22–24].

The type-I interferons consist of several members belonging to IFNα class and a single IFN-β. They act in a paracrine and autocrine manner on various immune and non-immune cells by binding to interferon receptors, IFNAR1 and IFNAR2. This binding triggers a cascade of downstream signalling events mediated by JAK1 and tyrosine kinase 2 (TYK2), which ultimately phosphorylate and activate STAT1 and STAT2. Activated STAT proteins assemble with the IRF9 to form an IFN-stimulated gene factor 3 (ISGF3) complex. STAT1/STAT1 can also form homodimers, known as IFNγ activating factor (GAF). Finally, ISGF3 and GAF bind to the ISREs and IFN-γ-activated sequence (GAS) elements, respectively, leading to the transcriptional activation of numerous ISGs, whose integrated action helps to achieve an antiviral state [25–28].

This study highlights the significant upregulation of hsa-miR-203a-3p expression upon RNA virus infection and poly(I:C) transfection and its impact on host antiviral responses. The hsa-miR-203a-3p binds to the 3’UTR of multiple transcripts belonging to IFNA and IFNA signalling pathway (IFNAR1, IFNA2, IFNA4, IFNA7, IFNA10, IFNA14, IFNA16, IFNA17, IFNA21). The ectopic expression of hsa-miR-203a-3p in HEK-293T, A549, and HeLa cells attenuates the type-I interferon-mediated antiviral responses, leading to an increase in viral load. To elucidate the cell-type specificity and function of miR-203a-3p, multiple cell lines representing distinct biological systems were used. A549 human lung epithelial cells served as the primary model, reflecting the respiratory epithelium targeted by influenza and other RNA viruses [29,30]. HEK-293T cells were used for dual luciferase reporter assays, as they offer excellent transfection efficiency and consistent expression of plasmids. In contrast, primary human peripheral blood mononuclear cells (hPBMCs) provided a physiologically relevant immune context. HeLa cells were also included as a non-respiratory epithelial model to determine if miR-203a-3p-mediated regulation extends beyond the respiratory epithelium. This experimental design enables a thorough evaluation of miR-203a-3p expression dynamics and potential regulatory roles during RNA virus infection across various cellular systems.

Material and methods

miRNA identification and pathway analysis

The NCBI-GEO database was queried for miRNA profiling experiments and databases relevant to H7N9, HCV, and DENV2 infections. GSE150782, GSE40744, and GSE135311 were selected based on experimental design and relevance to the study. Differentially expressed miRNAs were identified in control and infected samples of the given datasets using the GEO2R online tool [31,32]. TargetScan 7.1 [33,34] and miRabel [35] were used as default parameters to predict the target genes of selected miRNAs.

Cells, transfection, plasmids, mimics, and inhibitors

A549 human adenocarcinoma alveolar basal epithelial cells (Cell Repository, NCCS, India), HeLa cervical cancer cells (Cell Repository, NCCS, India), and HEK293T human embryonic kidney cells (ATCC, CRL-3216) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (Gibco). Cells were maintained at 37°C in a humidified incubator with 5% CO₂.

For transfection experiments, cells were seeded in 6-, 12-, or 24-well cell culture plates, depending on the experiment’s requirements. Transfections were carried out when cells reached 70–90% confluency. Plasmid, miRNA mimics (Invitrogen), inhibitors, and negative control or poly I:C were transfected according to the manufacturer’s protocol in HEK293T and HeLa cells using Lipofectamine 2000 (Invitrogen), and Lipofectamine 3000 (Invitrogen) was used for A549 cells, and respective samples were collected at desired time points.

Viruses and infection

Influenza virus (strain PR8, A/PR8/H1N1) and GFP-tagged NDV (strain LaSota) viruses were used for infection experiments. Viral stocks were prepared in pathogen-free embryonated chicken eggs and stored at −80°C. The multiplicity of infection (MOI) was calculated based on viral titre and cell density [36,37].

Cells were seeded in 6 or 12-well tissue culture plates at densities of 0.3x10⁶ or 0.1x10⁶ cells/well. 24 hours post-transfection, cells were washed with SF-DMEM or 1x Phosphate-buffered saline (PBS) and infected with NDV-GFP at 0.5 MOI and A/PR8/H1N1 at 0.5, 1, and 2 MOI. Virus suspensions in SF-DMEM were added to the cells and incubated for 1 hour at 37°C. Cells were then washed with SF-DMEM or 1x Phosphate-buffered saline (PBS), incubated in complete DMEM, and supplemented with 1% FBS. Replacement media for A/PR8/H1N1 infection was supplemented with L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (1 μg/ml). GFP-tagged NDV was kindly provided by Dr. Peter Palese, Icahn School of Medicine at Mount Sinai, New York, USA.

Luciferase reporter assay

Cells were seeded in 24-well plates and, after 24 hours, transfected with 150 ng/well UTR-luc-construct and 20 nM/well Negative control (NC)/m203a miRNA mimic or empty vector (EV)/pMIR-203a plasmid(p203). PRLTK was used as a control at a 20 ng/well concentration. After 24 hours, cells were lysed using a passive lysis buffer provided with the Dual-Glo Luciferase reagent assay kit.

Firefly luciferase luminescence was measured by mixing 50 µl of Dual-Glo Luciferase Reagent with 50 µl of cell lysate in an opaque 96-well plate. To measure the Renilla luciferase activity, an equal volume of Stop & Glow reagent was added per well. After 10 minutes of incubation, the luminescence of Renilla luciferase was measured. The luminescence intensity of Renilla luciferase was used to normalize the firefly luciferase luminescence. Luminescence signals were quantified using Promega GloMax multi-microplate reader

Quantitative real-time reverse transcription-PCR

Total RNA was extracted using TRIzol reagent (Invitrogen) and converted into cDNA using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s protocol. Reverse Transcription-quantitative PCR (RT-qPCR) was performed using gene-specific primers (sequences provided in supplementary table S1) using CYBR green chemistry (Bio-Rad) on the QuantStudio 3 Real-Time PCR system; 18S rRNA was used as an internal control for normalization. TaqMan-based RT-qPCR was conducted for miRNA expression analysis using a universal PCR master mix (Applied Biosystems) and specific TaqMan probes for U6 and hsa-miR-203a-3p. U6 served as the internal control for normalization.

Flow cytometry analysis

Cells were seeded in six-well plates and, at 60–70% confluency, transfected with pHsa-miR-203a-3p or pMIR-empty vector as a control. After 24 hours, cells were washed with phosphate-buffered saline (PBS) and infected with NDV-GFP at 1.5 MOI for 1 hour. Infected cells were washed with PBS, replenished with DMEM containing 1% FBS, and incubated overnight. Cells were detached using a 0.25% trypsin-EDTA solution, washed with PBS, and fixed with 4% paraformaldehyde for 5 minutes. Fixed cells were washed twice with PBS, resuspended in PBS, and analysed using a BD FACSAria II flow cytometer. FlowJo software (BD Biosciences) was used to analyse the data, and the fluorescence intensity was measured to quantify the levels of GFP expression.

RNA immunoprecipitation

RNA immunoprecipitation was performed as described in the previous articles [38,39]. HEK 293T cells were lysed in lysis buffer (0.5% NP-40, 150 mM KCl, 25 mM tris-glycine (pH 7.5) and incubated with M2 Flag affinity beads overnight at 4°C. Beads were washed 5 times with washing buffer (300 mM NaCl, 50 mM Tris-glycine [pH 7.5], 5 mM MgCl₂, 0.05% NP-40). Following the manufacturer’s protocol, Trizol reagent was used to get RNA from immunoprecipitated ribonucleoproteins (RNPs). The quality and quantity of extracted RNA were assessed using a NanoDrop spectrophotometer.

Measurement of viral titre

Madin-Darbin Canine Kidney (MDCK) cells were used for viral titre assays. Cells were infected with A/PR8/H1N1 or NDV in the serum-free MEM medium with viral supernatant. After 1 hour of virus infection, the cells were washed with 1X PBS and overlaid with 1.2% Avicel (Sigma-Aldrich; equivalent to Avicel RC-581 from FMC Corp.) in 2 mL maintenance medium. They were then incubated at 37°C with 5% CO₂ for 96 hours. Avicel overlays were removed, and the cells were fixed with 4% paraformaldehyde for 20 min. Fixed cells were stained with a 0.5% crystal violet solution, and plaques were counted under the microscope using transmitted light microscopy. For the 50% tissue culture infective dose (TCID50) assay, infected monolayers were fixed and stained with 1% crystal violet after 96 hours of infection. The TCID50 was then calculated based on the Reed and Muench method, as described previously [40,41].

Statistical analysis

All experiments were performed with appropriate control or mock-transfected samples and conducted independently at least two to three times. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were carried out using GraphPad Prism software to assess significance. Differences between the two groups were evaluated using an unpaired, two-tailed Student’s t-test. A p-value < 0.05 was considered statistically significant. The specific statistical tests applied to each dataset are indicated in the corresponding figure legends.

Results

Hsa-miR-203a-3p is induced during RNA virus infection

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression, including those involved in innate immunity. To identify novel microRNAs regulating antiviral innate immunity, we compared publicly available microarray data sets (GSE150782, GSE40744) and NGS data (GSE135311), which included different cells infected with H7N9, HCV, and DENV2. Dysregulated miRNAs were identified based on a p-value threshold of <0.05 and a log2 fold change greater than 0.5 or less than −0.5. Among them, hsa-miR-203a-3p, miR-378c, and miR-199a-5p were the only miRNAs significantly upregulated across all three datasets (Figure 1(A)). These three miRNAs were then analysed for their target genes using TargetScan 7.2 and miRabel. The predicted targets were further examined through BioPlanet, KEGG pathway, and Reactome analyses to understand the affected pathways [42–45]. Our findings indicate that hsa-miR-203a-3p plays a key role in regulating the Interferon alpha (IFNA) signalling pathway, TRAF6-mediated IRF7 activation, and the RIG-I-like receptor signalling pathway (Figure 1(B,C)), suggesting this miRNA is crucial during RNA virus infections. These pathways were selected based on top-ranked p-values and odds ratios (Fig. S1A-B), highlighting their strong association with the identified gene set. As hsa-miR-203a-3p targets antiviral pathways and is upregulated during RNA virus infection (Figure 1(D-F)), we decided to further study its role during RNA virus infection. To this end, first, we analysed the expression of miRNA-203a-3p in PBMC, A549, HEK293T and HeLa cells upon A/PR8/H1N1 or NDV infection and found that these viruses induced the expression of hsa-miR-203a-3p multifold (Figure 1(G-K)). Similarly, we observed the induction of hsa-miR-203a-3p upon transfection of A549 cells with poly(I:C), which activates retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) pathways (Fig. S1C). In contrast to hsa-miR-203a-3p, miRNA-378c and miR-199a-5p did not appear to target innate immune pathways (Fig. S2A – E). Collectively, these results suggest that RNA virus infection and mimicking RNA virus infection induce hsa-miR-203a-3p.

Figure 1.

Identification and pathway analysis of miR-203-3p: (A) microarray datasets for RNA virus infections, including H7N9, HCV and DENV2 were obtained from the gene expression omnibus (GEO) database and analysed using the GEO2R tool to identify differentially expressed miRNAs. Dysregulated miRNAs were selected based on a p-value < 0.05 and a log2 Fold change (FC) threshold of >0.5 or <−0.5. The analysis identified hsa-miR-203a-3p, miR-378c and miR-199a-5p were significantly upregulated across all three datasets. (B) predicted target genes were analyzed using Enrichr to identify significantly enriched pathways. A clustergram visualizes genes involved in the top-enriched pathways based on reactome pathway analysis. (C) pathway enrichment analysis highlights the most significant pathways associated with the miRNA target genes across multiple databases, including BioPlanet, KEGG, and reactome. (D-F) volcano plots depict the differential expression of hsa-miR-203a-3p in the analyzed datasets. (G-K) induction of hsa-miR-203a-3p in PBMCs, and HEK293T, HeLa, and A549 cell lines at indicated time by RT-qPCR after infection with A/PR8/H1N1 and NDV. Statistical significance is indicated as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Hsa-miR-203a-3p directly targets the 3’UTR of IFNA members and the IFNA signalling pathway

In silico analysis indicates that hsa-miR-203a-3p binds to the 3’UTR of the interferon signalling pathway genes such as IFNAR1, JAK1, and STAT1, including various sub-members of interferon alpha (IFNA1, IFNA2, IFNA4, IFNA7, IFNA10, IFNA14, IFNA16, IFNA17) (Figure 2(A)). IFNA is a predominant member of type-I interferon and a key contributor to the antiviral response. To investigate the IFNA members being targeted by hsa-miR-203a-3p, we cloned full-length UTRs of these genes downstream of the luciferase reporter gene in the pMIR-REPORT vector. HEK293T cells were co-transfected with UTR-cloned luciferase reporter plasmids along with NC or m203a. Cells were lysed and subjected to luciferase assay after 48 hours of transfection. As expected, overexpression of hsa-miR-203a-3p significantly reduced the luciferase activity of IFNAR1, IFNA1, 4, 7, and 16, JAK1, STAT1, and SOCS3 (Figure 2(B-I)), indicating that hsa-miR-203a-3p binds to the 3’UTR of these genes, resulting in the downregulation of luciferase activity. However, luciferase activity did not change when the IRF7 UTR lacking hsa-miR-203a-3p target sequence was co-transfected with miR203-3p (Fig. S3A).

Figure 2.

Hsa-miR-203a-3p directly targets the 3’UTR of IFNA members and it’s signalling pathway genes: (A) schematic representation of conserved region in genes that are predicted to be targeted by hsa-miR-203a-3p, in the various members of type I interferon (IFN) and type I IFN signalling pathway. (B-I) the 3' UTRs of IFNAR1, IFNA1, IFNA4, IFNA7, IFNA16, JAK1, STAT1, and SOCS3, were cloned into the pMIR-report luciferase vector and co-transfected with either NC or m203. HEK293T cells were transfected with plasmids containing IFNAR1-luc, IFNA1-luc, IFNA4-luc, IFNA7-luc, IFNA16-luc, JAK1-luc, STAT1-luc, or SOCS3-luc, along with 20 ng of pRL-TK (renilla luciferase for normalization), 100 ng of full-length wild-type (WT) 3' UTR, and either 350 ng of the hsa-miR-203a-3p expression plasmid or 20 nM of a NC/hsa-miR-203a-3p mimic. After 48 hours, cells were lysed, and luciferase activity was measured. (J-Q) the hsa-miR-203a-3p binding sites in the IFNAR1, IFNA1, IFNA4, IFNA7, IFNA16, JAK1, STAT1, and SOCS3 3' UTRs were mutated using site-directed mutagenesis (SDM). The miRNA binding site sequence CATTTCA was mutated to ACTTGAC, and luciferase assay was performed in similar method as above as indicated constructs. The level of significance is indicated as follows: * (p < 0.01), ** (p < 0.001), *** (p < 0.0001), and **** (p < 0.00001). Assay. Statistical.

To further validate the hsa-miR-203a-3p targeting sites in the 3’UTRs of target genes, the binding site were mutated from CATTTCA to ACTTGAC in the 3’UTRs of the IFNAR1, IFNA1, IFNA4, IFNA7, IFNA16, JAK1, STAT1, and SOCS3 genes using Site-Directed Mutagenesis (SDM). After altering the miRNA binding site, the luciferase assay was performed as described previously. Luciferase assays with mutated constructs showed comparable luciferase activity with empty vector upon hsa-miR-203a-3p mimic transfection, confirming the specificity of hsa-miR-203a-3p’s interaction with the 3’UTR of target genes (Figure 2(J-Q)).

Together, these results suggest that hsa-miR-203a-3p directly binds to the 3’UTRs of IFNA subtypes, IFNAR1, JAK1, STAT1, and SOCS3, leading to suppression of target gene transcript and may result in the downregulation of type I IFN and type I IFN signalling pathway.

Hsa-miR-203a-3p target genes through RISC

MicroRNAs target transcripts through a multiprotein complex known as the RNA-induced Silencing Complex (RISC), and Ago2 is one of the essential components of RISC. To determine how hsa-miR-203a-3p regulates targeted transcripts. RNA immunoprecipitation (RIP) assay was performed along with Ago2. HEK293T cells were transfected with FLAG-tagged Ago2 and hsa-miR-203a-3p mimic (m203) or negative control (NC), followed by NDV or A/PR8/H1N1 infection (Figure 3(A)). Twenty-four hours post-infection, RNA associated with Ago2 was immunoprecipitated using FLAG antibody. Subsequently, the levels of Pan-α, IFNAR1, IFNA1, JAK1, and STATI transcripts were measured. The expression of these transcripts was significantly enriched in samples where hsa-miR-203a-3p was introduced compared to the negative control (NC). The overexpression of hsa-miR-203a-3p in cells was confirmed, as shown in Figure 3(B). These results indicate the direct interactions between hsa-miR-203a-3p and Pan-α, IFNAR1, IFNA1, JAK1, and STATI mRNA transcripts (Figure 3(C-G)). Here, IRF7 was used as a non-target control (Fig. S3B).

Figure 3.

Hsa-miR-203a-3p binds with type I IFN pathway genes through Ago2-mediated RISC: (A) schematic representation of the RNA immunoprecipitation (RIP) assay. HEK293T cells were either left untransfected or transfected with a flag- Ago2 plasmid along with either a negative control (NC) or hsa-miR-203a-3p (m203) mimic. Following transfection, cells were infected with A/PR8/H1N1 or NDV at an MOI of 1.5 for 24 hours. Cell lysates were subjected to Ago2-FLAG capturing using an anti-flag antibody. (B) the level of hsa-miR-203a-3p associated with the RNA-induced silencing complex (RISC) was quantified. (C-G) the mRNA levels of pan-IFNA, IFNAR1, IFNA1, JAK1, and STAT1 in immunoprecipitated samples were measured using RT-qPCR. (H-I) A549 cells were transfected with NC, m203, or an inhibitor of hsa-miR-203a-3p (I203), followed by A/PR8/H1N1 infection. After 24 hours, cells were trypsinized, and protein lysates were collected for Western blot analysis. The protein levels of JAK1 and IFNAR1 were assessed using anti-JAK1 and anti-IFNAR1 antibodies. (J-K) A549 cells were transfected with NC, m203, or I203. After 24 hours, cells were infected with A/PR8/H1N1, and supernatants were collected 24 hours post-infection. And measured the IL-6 and IFN-β levels. By ELISA. Statistical significance is indicated as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Furthermore, these observations were confirmed at the protein level by Western blot analysis using JAK1 or IFNAR1-specific antibodies. Both JAK1 and IFNAR1 play a pivotal role in enhancing type I IFN production through a positive feedback loop. As demonstrated above in RIP assay, the relative enrichment of these transcripts was high. Therefore, we analysed the protein of JAK1 and IFNAR1. As shown in Figure 3(H,I), hsa-miR-203a-3p mimic (m203) significantly reduced the protein of IFNAR1 and JAK1 compared to the NC-transfected cells, whereas treatment with the hsa-miR-203a-3p inhibitor, which sequesters endogenous hsa-miR-203a-3p, modestly enhanced the expression of both IFNAR1 and JAK1 (Figure 3(H,I)). Additionally, the effect of hsa-miR-203a-3p on IL-6 and IFN-β production in the culture supernatant was measured using ELISA. Notably, IL-6 production is driven by NF-κB, which is influenced by type I interferons. Similarly, IFN-β production is regulated through a positive feedback loop involving both IFN-α and IFN-β. To this end, A549 cells were transfected with NC, hsa-miR-203a-3p mimic (m203), or hsa-miR-203a-3p inhibitor (I203), followed by infection with A/PR8/H1N1 after 24 hours; the supernatant was collected 24 hours post-infection, and IL-6 protein levels were quantified using ELISA. As shown in Figure 3(J), the production of IL-6 was significantly reduced compared to the NC, whereas I203 transfection modestly enhanced IL-6 production. Next, IFN-β production was also measured by ELISA. Cells transfected with hsa-miR-203a-3p showed a marked reduction of IFN-β compared to the NC-transfected cells, whereas introduction of I203 showed comparable levels of IFN-β with NC-transfected cells. (Figure 3(K)), suggesting a broader regulatory role of hsa-miR-203a-3p in cytokine responses. Altogether, these results indicate that hsa-miR-203a-3p directly interacts with IFNA family genes and IFNAR1-JAK1-STAT1 signalling axis and suppresses at both the RNA and protein levels, thereby modulating type I IFN signalling.

To further validate the functional relevance of the IFNAR1–JAK1–STAT1 signalling axis, type I interferon-deficient Vero E6 cells were transfected with either Pmir-EV (empty vector control) or Pmir-miR-203a-3p. After 24 hours, the cells were infected with A/PR8/H1N1 or Newcastle disease virus (NDV) at a multiplicity of infection (MOI) of 1.5. Viral RNA levels were measured 24 hours post-infection using RT-qPCR. The results showed an increase in viral load in miR-203a-3p-transfected cells (Fig. S4A-B). These findings suggest that in addition to hsa-miR-203a-3p also targets JAK1 and STAT1 transcripts, thereby suppressing ISG activation and promoting viral replication.

Hsa-miR-203a-3p suppresses antiviral responses

Viral infections are known to trigger the upregulation of innate immune cytokines, particularly type-I interferons such as IFN-α and IFN-β, which are essential for activating interferon-stimulating genes (ISGs) that collectively inhibit viral replication and enhance immune responses. To assess and validate the impact of hsa-miR-203a-3p on type-I interferon (IFN) expression, A549 cells were transfected with negative control (NC), hsa-miR-203a-3p mimic (m203), or hsa-miR-203a-3p inhibitor (I203), followed by infection with A/PR8/H1N1 or NDV. We observed that hsa-miR-203a-3p mimic significantly reduced IFN-α and IFN-β transcripts at various time points post-infection with both A/PR8/H1N1 and NDV in A549 cells and hPBMCs (Figure 4(A-D)). Conversely, treatment with the hsa-miR-203a-3p inhibitor (I203) restored IFN-α and IFN-β expression (Figure 4(A-D)). To investigate the downstream effect of reduced expression of Type I interferons (IFN-α and IFN-β), we analysed the activation of the Interferon Stimulated Response Element (ISRE), a promoter element critical for ISG activation. Using an ISRE luciferase reporter (pISRE-luc) plasmid containing the ISRE promoter present in the ISGs. The ISRE sequence cloned upstream of a luciferase gene, HEK293T cells were transfected with either a negative control (NC) or m203a-3p, along with pISRE-Luc. After 24 hours, the cells were either left untreated or treated with IFN-β for 24 hours. The results revealed a significant reduction in luciferase activity in cells transfected with hsa-miR-203a-3p, whereas no significant difference was observed between cells transfected with the ISRE construct alone and those co-transfected with ISRE and a negative control (NC) (Figure 4(E)). These observations suggest that hsa-miR-203a-3p suppresses Type I interferon (IFN) signalling, thereby diminishing the expression of IFN-α, IFN-β, and downstream interferon-stimulated genes (ISGs).

Figure 4.

Hsa-miR-203a-3p overexpression suppresses type-I IFN and type-I IFN-inducible responses: (A) A549 cells were transfected with a hsa-miR-203a-3p mimic (m203a), a negative control (NC), or a hsa-miR-203a-3p inhibitor (I203) at a concentration of 50 nM. After 24 hours, cells were infected with A/PR8/H1N1 (MOI = 1.5). Samples were collected at 12 and 48 hours post-infection to measure IFNα expression by RT-qPCR. (B – C) similarly, IFN-β expression was measured at 12, 24, and 48 hours following A/PR8/H1N1 or NDV infection. (D) the level of IFN-β was also measured in peripheral blood mononuclear cells (PBMCs), which were transfected with NC, m203a or I203a at 50 nM followed by A/PR8/H1N1 infection. The relative expression of IFN-β was measured by RT-qPCR at 24, and 48 hours post-infection. (E) HEK293T cells were transfected with either pISRE alone, pISRE + m20a, or pISRE + negative control (NC). After 24 hours, the cells were stimulated for 24 hours with IFN-β, and luciferase activity was measured. Statistical significance is indicated as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Hsa-miR-203a-3p promotes the RNA virus replication

To determine the effect of hsa-miR-203a-3p on viral load, A549 cells were transfected with negative control (NC), hsa-miR-203a-3p mimic (m203), or m203 inhibitor (I203). Subsequently, the transfected cells were infected with A/PR8/H1N1 or Newcastle Disease Virus (NDV) at a multiplicity of infection (MOI) of 1.5. The viral load was then quantified at 12, 24, and 48 hours post-infection by quantitative PCR. Expression of the A/PR8/H1N1 NP gene (for influenza) and the NDV L-pol gene, which encodes the large polymerase of NDV, was also measured. A significant increase in viral load was observed in cells transfected with the hsa-miR-203a-3p mimic (m203) compared to the negative control, while the viral load was reduced in cells treated with the hsa-miR-203a-3p inhibitor (I203) (Figure 5(A-B)). To further validate our observation, the viral load in A549 cells was determined by cytofluorometric analysis of cells infected with GFP-tagged NDV using flow cytometry. Samples included cells with negative control + NDV-GFP, m203 + NDV-GFP, and I203 + NDV-GFP. hsa-miR-203a-3p mimic treatment resulted in an increased NDV load, while the hsa-miR-203a-3p inhibitor (I203) led to a decrease in viral load (Figure 5(C-D)). Similar results were observed in human peripheral blood mononuclear cells (hPBMCs) at 24 and 48 hours post-infection, further supporting the role of hsa-miR-203a-3p in enhancing the viral replication (Figure 5(E)). We also investigated whether hsa-miR-203a-3p affects viral entry or restricts viral replication during the early stages of infection. We transfected A549 cells with either a negative control (NC) or hsa-miR-203a-3p mimic (m203). After 24 hours of transfection, the cells were infected with PR8, and samples were collected at 1, 3 and 12 hours post-infection. The viral load was quantified using RT-qPCR, which showed an increase in viral load at early time points post-infection. This indicates that hsa-miR-203a-3p does not interfere with viral entry or the early stages of infection (Fig. S5A). Altogether, these results suggest that overexpression of hsa-miR-203a-3p enhances viral replication by suppressing type I interferon genes, which are essential for antiviral defence. This finding provides valuable insight into the regulatory role of hsa-miR-203a-3p in host-pathogen interactions and highlights its potential as a therapeutic target for controlling viral infections.

Figure 5.

Hsa-miR-203a-3p enhances RNA virus replication: (A-B) A549 cells were transfected with a hsa-miR-203a-3p mimic (m203a), a negative control (NC), or a hsa-miR-203a-3p inhibitor (I203) at a concentration of 50 nM. After 24 hours, cells were infected with A/PR8/H1N1 (PR8) or Newcastle disease virus (NDV) at a multiplicity of infection (MOI) of 1.5. Samples were collected at 12, 24, and 48 hours post-infection. (A) the relative expression of the NP gene was quantified by RT-qPCR. (B) the expression of the NDV L polymerase (L pol) gene was also measured at the same time points. (C-D) fluorescence-activated cell sorting (FACS) analysis was performed to measure viral load. A549 cells were transfected with NC, m203a, or I203 at 50 nM. After 24 hours, cells were infected with NDV (MOI = 1.5) and incubated for an additional 24 hours. Cells were then trypsinized, washed with 1× PBS, fixed with 4% formaldehyde, and analyzed by flow cytometry for GFP. (E) a similar experiment was conducted using peripheral blood mononuclear cells (PBMCs) transfected with NC, m203a or I203a at 50 nM, followed by infection with A/PR8/H1N1. The relative expression of the NP gene was measured by RT-qPCR at 24 and 48 hours post-infection. Statistical significance is indicated as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and****(p < 0.0001).

Discussion

The innate immune system relies on various pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and cytosolic DNA sensors [46,47]. The RNA viruses are primarily detected by RIG-I, MDA5, TLR3, TLR7, and TLR8 that recognize the single-stranded RNA, 5'-triphosphate RNA, or double-stranded RNA. Additionally, the NLRs (NLRP3) and DExD/H helicases (DDX3) also contribute to virus recognition by sensing the RNA-induced stress signals and viral RNA intermediates, respectively [48]. Activation of these sensors initiates a robust type-I interferon and pro-inflammatory cytokine response, which ultimately upregulates the expression of several innate immune genes and non-coding RNAs (ncRNAs) that collectively shape the antiviral immunity [49]. Recent reports suggest that ncRNAs, such as miRNAs, long non-coding RNAs, and circular RNAs, play a pivotal role in fine-tuning the antiviral response by regulating gene expression, acting as decoys for RNA sensors, or sponging miRNAs [50].

miRNAs can bind to the 3’-UTRs of their target transcripts, leading to the silencing of host and viral transcripts primarily through translational repression and mRNA degradation mechanisms. In this study, we found that the expression of hsa-miR-203a-3p was significantly elevated upon infection with RNA viruses, such as NDV and A/PR8/H1N1, as well as upon transfection with dsRNA analogue polyinosinic-polycytidylic acid (I:C). Notably, the microarray and NGS data from three independent studies available at the NCBI-GEO database also corroborate that hsa-miR-203a-3p is a consistently upregulated microRNA during RNA virus infections, further supporting its potential role as a key regulator in host antiviral defence mechanisms. Computational analysis revealed that hsa-miR-203a-3p potentially targets multiple key components of the interferon signalling pathway, including members of the IFNA family, IFNAR1, JAK1, STAT1, and SOCS3. The same interactions were further validated through luciferase reporter assays. Furthermore, the luciferase assays with a mutated 3’UTR of target genes showed comparable luciferase activity with the empty vector, demonstrating the specific binding of hsa-miR-203a-3p to the 3’UTRs of these genes. Finally, direct biochemical evidence of the interaction between hsa-miR-203a-3p and predicted target transcripts was provided by the RNA immunoprecipitation (RIP) assay, which captures miRNA and its target RNAs in association with Ago2, a vital component of the RNA-induced silencing complex (RISC). RIP captures endogenous miRNA-RISC complexes to test whether a specific miRNA and a candidate RNA physically associate in cells. These findings were further corroborated by western blot analysis, which showed decreased levels of JAK1 and IFNAR1 upon overexpression of hsa-miR-203a-3p, further confirming its regulatory effect [51,52].

We explored the functional implications of hsa-miR-203a-3p for viral replication by overexpressing or inhibiting the endogenous hsa-miR-203a-3p. The results demonstrated that overexpression of hsa-miR-203a-3p supports viral replication, as shown by increased levels of the A/PR8/H1N1 NP and NDV L-pol genes. Conversely, inhibiting hsa-miR-203a-3p led to reduced viral loads, underscoring its role as a positive regulator of viral replication. These observations were consistent across different cell lines and human primary cells, i.e. peripheral blood mononuclear cells (PBMCs), reinforcing the universality of this mechanism. Furthermore, the suppression of type I interferon signalling by hsa-miR-203a-3p was demonstrated by a significant decrease in the levels of IFN-β and IFN-α production in cells transfected with hsa-miR-203a-3p mimics during viral infection. This suppression likely led to the observed increase in viral replication (Fig. 6), as type I interferons are essential for mounting an effective antiviral response. The restoration of IFN-α and IFN-β expression following the inhibition of hsa-miR-203a-3p highlights its significance in antiviral immunity and immune homoeostasis. Further research will deepen our understanding of hsa-miR-203a-3p and aid in developing innovative therapeutic strategies against RNA virus infections and complex autoimmune diseases such as SLE. A preliminary version of this study has been published as a preprint [53].

Funding Statement

This research was supported by the Indian Institute of Science Education and Research Bhopal (IISER Bhopal). The institute had no role in the design of the study, data collection, analysis, interpretation, or manuscript writing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contribution

P.K., A.K., Ak.K., and H.K. conceptualized the study. P.K. and A.K. performed experiments. A.K. analysed data from the repository, and P.K. and A.K. did experiments on A/PR8/H1N11 and NDV in A549, 293T, HeLa, and PBMC cells. P.K., Ak.K., and H.K. prepared the manuscript; Kunal Arora helped with proofreading and editing, and H.K. supervised the entire project.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [H.K], upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2026.2640211