Therapeutic role of miR-19a/b protection from influenza virus infection in patients with coronary heart disease

Patients with pre-existing medical conditions are at a heightened risk of contracting severe acute respiratory syndrome (SARS), SARS-CoV-2, and influenza viruses, which can result in more severe disease progression and increased mortality rates. Nevertheless, the molecular mechanism behind this phenomenon remained largely unidentified. Here, we found that microRNA-19a/b (miR-19a/b), which is a constituent of the miR-17-92 cluster, exhibits reduced expression levels in patients with coronary heart disease in comparison to healthy individuals. The downregulation of miR-19a/b has been observed to facilitate the replication of influenza A virus (IAV). miR-19a/b can effectively inhibit IAV replication by targeting and reducing the expression of SOCS1, as observed in cell-based and coronary heart disease mouse models. This mechanism leads to the alleviation of the inhibitory effect of SOCS1 on the interferon (IFN)/JAK/STAT signaling pathway. The results indicate that the IAV employs a unique approach to inhibit the host’s type I IFN-mediated antiviral immune responses by decreasing miR-19a/b. These findings provide additional insights into the underlying mechanisms of susceptibility to flu in patients with coronary heart disease. miR-19a/b can be considered as a preventative/therapy strategy for patients with coronary heart disease against influenza virus infection.


INTRODUCTION
The COVID-19 pandemic has resulted in unparalleled worldwide disruption.The seasonal cycle of various infectious diseases, including influenza, has been disrupted globally since early 2020 due to changes in exposure patterns and mobility (Figure S1).Influenza A virus (IAV) is a major and annual epidemic respiratory pathogen associated with serious economic and health issues. 1It belongs to the Orthomyxoviridae family of negative-strand RNA viruses, which is mainly divided into types A, B, C, and D. The high pathogenicity and variability of IAV can significantly increase its potential to cause severe respiratory infections as well as widespread epidemics or influenza, 2,3 which seriously endanger human well-being.The 1918 flu (H1N1) killed nearly 50 million people, 4 and the 2009 H1N1 pandemic killed nearly 17,000 people. 5,6Though H1N1 pdm09 has been included in the influenza vaccine since 2010, it still propagates in the community annually and was the predominant IAV strain during the 2019-2020 influenza epidemic. 7Influenza infection is associated with increased mortality in patients with underlying conditions such as cardiovascular disease, chronic kidney disease, diabetes, chronic respiratory disease, and a range of other chronic conditions. 8he causative mechanism behind adverse outcomes of underlying conditions in patients with influenza was largely unknown.
Coronary heart disease (CHD) is a common disease in middle-aged and elderly people, and the age of its outbreak tends to be younger. 9tudies have confirmed that high blood pressure, high blood glucose, high blood lipid, smoking, drinking, and genetic factors are intensively related to the risk factors of CHD. 10 Patients with cardiovascular disease have reduced baseline cardiopulmonary and renal function, increasing the risk of respiratory disease.Madjid et al. have demonstrated a clear association between influenza epidemics and the increased rate of deaths from CHD. 11 Several mechanisms have been revealed that show that influenza can increase the risk of cardiovascular events, such as the activation of coagulation cascade, pro-inflammatory mediators, and sympathetic stimulation. 12However, the specific molecular mechanism through which CHD increased susceptibility to influenza virus remains largely unknown.
MicroRNAs (miRNAs or miRs) are short, single-stranded, non-coding RNAs composed of 21-23 nt that regulate the expression of target genes at the post-transcriptional level by impeding the translation or promoting the degradation of target mRNAs.Expression of virusderived miRNAs or modulation of host-derived miRNAs has been demonstrated as an effective strategy for viral immune evasion. 13,14onversely, host-derived miRNAs are responsible for the antiviral responses by the direct regulation of virus-derived nucleotides (vcRNA, vmRNA, or vDNA) stability or the modulation of innate and adaptive immunity. 146][17] For example, miR-34a can promote influenza virus-mediated apoptosis by binding to BAX16, while miR-let-7c can target and inhibit M1 expression of H1N1 influenza virus. 18Tambyah et al. observed significant expression changes in 193 miRNAs among 50 patients with severe H1N1 or H3N2 influenza infection. 19We hypothesized that miRNA expression differences in patients with CHD may exacerbate complications following influenza infection.
The miR-17-92 cluster is polycistronic and situated in the 13q31 region of the human chromosome.Its initial discovery as an oncogene was attributed to its ability to induce transformation. 20A single transcript transcribes six distinct miRNAs, namely miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a.It should be emphasized that despite miR-17-92 being transcribed as a singular transcript, there is a variance in the expression of individual miRNAs due to post-transcriptional processing. 21Studies indicate that the miR-17-92 gene cluster may play a role in the onset and progression of cardiovascular diseases.Mayer et al. 22 reported that reduced miR-19a expression in the bloodstream is linked to higher mortality rates in individuals with stable coronary artery disease (SCAD).The diminished expression of miR-19a in patients with SCAD may increase the likelihood of mortality due to cardiovascular disease.Singh and colleagues 23 discovered a correlation between reduced plasma miR-19b expression levels in individuals with acute coronary syndrome and an increased likelihood of aspirin resistance and major cardiovascular and cerebrovascular adverse events, including myocardial infarction and stroke.The limited investigation of miR-19 in influenza prompts us to hypothesize that its low expression in individuals with CHD may contribute to the development of severe influenza following infection.
The study revealed a decrease in the expression of miR-19a/b among patients diagnosed with CHD.Further analysis of in vivo and in vitro infection with IAV revealed that overexpression of miR-19a/b impeded SOCS1, consequently suppressing the release of inflammatory mediators.This observation may offer novel insights into the heightened susceptibility of patients with CHD to influenza.

Patients or mice with CHD exhibit low expression of miR-19a and miR-19b
Our study aimed to evaluate the predictive potential of circulating miRNAs in patients with CHD.We recruited 15 patients with CHD and 8 healthy people who had not received antitumor drugs, radiotherapy, immunosuppressants, hypoglycemic drugs, lipid-lowering drugs, vitamin C, aspirin, or antioxidant drugs prior to blood collection, and the work performed was accepted by the research ethics committee.Figure 1A displays the data of the patients with CHD and of the healthy people.Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll lymphocyte separation medium, and total RNA was extracted from collected blood samples.RT-qPCR was used to quantify miRNA expression in patients with CHD (Figure S2A).The results indicated lower expression of six members of miR17-92 in these patients compared to healthy individuals, with miR-19a and miR-19b showing the most significant decreases (Figure 1B).To assess the consistency of miR-19a and miR-19b in animals, we created a mice model with CHD.ApoE À/À mice were subjected to an 18-week highfat diet (Figure 1C), resulting in increased body size and weight compared to normal mice.Cardiac contractile ability was assessed using B-ultrasound, while lipid changes were measured using a test kit (Figures 1D and 1E).The results were similar to those of patients with CHD (Figure S2B).miRNA expression profiles were obtained from the lungs of ApoE À/À mice using transcriptome sequencing and bioinformatics techniques, resulting in the identification of 9 differentially expressed miRNAs (Figure 1F).The expression difference of miR-19a and miR-19b was observed in both PBMCs and lung tissues of ApoE À/À mice and normal mice, as depicted in Figure 1G.Axon guidance and focal adhesion were the most enriched signaling pathways, as indicated by the KEGG pathway analysis (Figure 1H).The Gene Ontology (GO) enrichment analysis indicated that intracellular components and membrane-bound organelles were the most pertinent target genes of differentially expressed miRNAs in cell components.Molecular functions primarily encompass protein binding, catalytic activity, and other related functions.At the biological level, the focus is primarily on cellular processes, biological regulation, and metabolic processes (Figure S2C).
The miR-19a/b complex exhibits in vivo attenuation of IAV replication Our hypothesis posits that miR-19a and miR-19b may play a role in the regulation of influenza virus.To confirm this conjecture, we sought to artificially increase the expression of miR-19a and miR-19b in ApoE À/À mice.miR-19a and miR-19b exhibit a high degree of functional similarity, as they share the same seed sequence (Figure S3A), differing by only one base, 24 so we conducted an experiment to investigate both miRNAs together.This study investigated the effect of an intravenous agomir-miR-19a/b mixture on susceptibility to influenza in ApoE À/À mice; the results are presented in a flow diagram (Figure 2A) and show that the expression levels of miR-19a/b in the PBMCs and lungs of ApoE À/À +agomir-NC-treated mice were lower than in NC mice.However, ApoE À/À +agomir-miR-19a/b-treated mice group showed higher expression levels of miR-19a/b than the ApoE À/À +agomir-NC-treated mice group, with significant differences (p < 0.05) (Figures 2B, 2C, and S3B).Subsequently, an investigation was conducted to determine the potential impact of miR-19a/b on the mortality and morbidity of IAVs in vivo.The wild animal disease research group at the Institute of Zoology, Chinese Academy of Sciences, isolated and preserved the BJ05/H1N1 subtype influenza virus during their previous monitoring efforts.The NC mice, ApoE À/À +agomir-NC-treated mice, and ApoE À/À +agomir-miR-19a/b-treated mice were intranasally inoculated with BJ05/H1N1 influenza virus at a dosage of 10 4 plaque-forming units.According to the findings, the administration of agomir-miR-19a/b to ApoE À/À mice effectively suppressed weight loss in the presence of BJ05/H1N1 influenza virus infection.On the contrary, subjects who did not receive agomir-miR-19a/b for ApoE À/À mice exhibited elevated levels of weight loss and more severe clinical symptoms, including curling, lethargy, dyspnea, and anorexia.Despite the eventual mortality of all mice inoculated with BJ05/H1N1, the administration of agomir-miR-19a/b injection resulted in a reduction of mortality in ApoE À/À mice and an increase in their survival time, as depicted in Figures 2D and  2E.Collectively, these findings suggest that miR-19a/b may ameliorate influenza virus infection symptoms in ApoE À/À mice.

Pathological data pertaining to mice infected with H1N1
In order to gain insight into the pathological characteristics of BJ05/ H1N1-infected groups, mice were euthanized at specific time points, including days 1, 2, 3, and 5 post-infection.The observed pathological changes in the NC mice, ApoE À/À +agomir-NC-treated mice, and ApoE À/À +agomir-miR-19a/b-treated mice primarily manifested in the lung tissues, characterized by pulmonary hemorrhage and pulmonary edema.The brain, intestine, spleen, and liver did not exhibit apparent lesions.However, the presence of infectious virus particles and viral RNA (vcRNA or vmRNA) was observed (Figure S4).The lung tissue's pathological changes in mice were observed to have significantly worsened over the course of the infection.The pulmonary hemorrhage area was observed to have expanded, and the level of pulmonary edema was found to have escalated.The pulmonary pathological alterations in three cohorts of mice were most severe at the 7-day mark following infection (Figure 3A).The ApoE À/À +agomir-NCtreated mice group exhibited more pronounced pathological changes compared to those in the ApoE À/À +agomir-miR-19a/b-treated group (Figure 3B).The ApoE À/À +agomir-NC-treated mice group exhibited pulmonary interstitial hyperplasia, alveolar structure disappearance, inflammatory cell infiltration, and significant pathological alterations in the lungs.The ApoE À/À +agomir-miR-19a/b-treated mice developed lung parenchyma lesions after 5 days of infection, whereas ApoE À/À +agomir-NC-treated mice showed an expansion of lesion area that persisted until 7 days post-infection (Figure 3B).We further examined the distribution of viral nucleoprotein in lungs.Results showed that virus particles were widely distributed in lung tissues of ApoE À/À +agomir-NC-treated mice, while the distribution of virus particles was limited in the ApoE À/À +agomir-miR-19a/b-treated group (Figures 3C and 3D).The distribution of viral nucleoprotein in lungs was further analyzed.The study found that virus particles were prevalent in the lung tissues of ApoE À/À +agomir-NC-treated mice, but were restricted in ApoE À/À +agomir-miR-19a/b-treated mice (Figures 3C and 3D).Mice injected with agomir-miR-19a/b exhibited greater resistance to BJ05/H1N1 infection compared to mice with CHD.This suggests that miR-19a/b may impede the replication of influenza virus.In general, the resistance of ApoE À/À mice injected with agomir-miR-19a/b to BJ05/H1N1 infection was stronger than that of ApoE À/À +agomir-NC-treated mice, indicating that miR-19a/ b may hamper the replication of influenza virus.Moreover, the extent of the lesions increased with the duration of infection.
The present study delved deeper into the expression of interferon g (IFN-g), tumor necrosis factor a (TNF-a), interleukin-1b (IL-1b), and IL-6, which are known to exert a crucial inhibitory effect on virus replication.The study findings indicate that the mice treated agomir-miR-19a/b exhibited elevated levels of IFN-g compared to the mice with CHD, as illustrated in Figure 3E.Furthermore, the significant pro-inflammatory factors IL-1b and IL-6 exhibited an increase in mice with CHD.An increase in TNF-a expression was noted in both ApoE À/À +agomir-NC-treated mice and ApoE À/À +agomir-miR-19a/b-treated mice.However, the rate of TNF-a upregulation in ApoE À/À +agomir-miR-19a/b-treated mice was comparatively slower than in CHD mice.The findings indicate that ApoE À/À mice treated with agomir-miR-19a/b may elicit antiviral responses more effectively than ApoE À/À +agomir-NC-treated mice.

miR-19a/b suppresses influenza virus replication
Following the successful demonstration of the ability of miR-19a/b to decrease mortality and morbidity in ApoE À/À mice infected with influenza in vivo, our subsequent inquiry focused on the potential impact of miR-19a/b on the inhibition of IAV replication in vitro.In order to achieve this objective, A549 cells were transfected with varying concentrations of agomir-miR19-a/b and subsequently exposed to BJ05/ H1N1 infection at multiplicity of infection (MOI) = 1.The replication of the virus was monitored at 24 h and 48 h post-infection (hpi).The findings indicate that miR-19a/b, at varying concentrations, effectively inhibited BJ05/H1N1 replication at both 24 and 48 hpi.Notably, the most potent inhibition of BJ05/H1N1 replication was observed with the use of 20 nM agomir-miR-19a/b, as depicted in Figure 4A.Transfection efficiency is shown in Figure S9A.In order to investigate the influence of miR-19a/b on influenza virus infection, we conducted an assessment of virus infection utilizing agomir-miR-19a/b.According to the data, the agomir-miR-19a/b exhibited a significant inhibitory effect on the expression of viral NP and M1 (Figure 4B).Transfection efficiency is shown in Figure S9B.Subsequent research results showed that miR19a/b has the ability to inhibit the replication of BJ05/H1N1 in A549 cells.
Several studies have examined miRNA expression following infection with IAV.The expression of miR-30 family members, specifically miR-30, has been observed to be regulated by IAV attack. 25Subsequently, we conducted an assessment to determine if IAV infection can regulate miR-19a/b in relevant cellular models.To this end, we assessed the expression levels of miR-19a/b in A549 cells at various time intervals following BJ05/H1N1 infection.A notable observation was made regarding viral infection, particularly in the initial phase of infection.The expression of miR-19a/b was observed to be significantly reduced in A549 cells that were infected with BJ05/H1N1.As the innate antiviral immune response takes place during the early stages of IAV infection, it is hypothesized that miR-19a/b may modulate influenza infection by regulating the innate antiviral immune response.The levels of miR-19a/b were measured in A549 cells that were infected with varying MOIs of BJ05/H1N1.The results indicate that the expression of miR-19a/b decreased in a dose-dependent manner in A549 cells infected with BJ05/H1N1, as shown in Figures 4C and 4D.We used another virus, NDV1/Pigeon/BJ/2023, to infect DF1 cells 24 hpi and obtained similar results in Figure S5.
Collectively, these findings indicate that miR-19a and miR-19b exerted inhibitory effects on influenza virus replication at the cellular level.
The suppressor of cytokine signaling proteins 1 has been identified as a target of miR-19a/b To investigate the mechanism by which miR-19a/b inhibits influenza replication, we identified their potential targets.RNA hybrid sequence analyses suggest that the miR-17-92 cluster does not directly target IAV viral genomes.As a result, we propose that miR-17-92 may regulate IAV replication by modulating the cellular signaling pathway.The TargetScan 7.2 and miRBase databases were utilized for identifying potential targets in bioinformatics analysis (Figure 5A).SOCS1, which has potential binding sites, is a significant target involved in the antiviral response (Figure 5B).miR-19a/b was identified as a potential target in the 3 0 UTR region of SOCS1 mRNA through comparison analysis, and we analyzed the binding site UUUGCACA between miR-19a/b and the SOCS1 mRNA 3 0 UTR region through NCBI.Wild-type (WT)-SOCS1 and mutated (MUT)-SOCS1 were designed and evaluated by dual-luciferase reporter experiment.The results showed that the pmirGLO-SOCS1-WT+ miR-19a/b group had the lowest firefly/Renilla luciferase expression, whereas the binding site MUT-SOCS1 did not downregulate firefly/ Renilla luciferase expression.It was confirmed that miR-19a/b inhibited SOCS1 expression by targeting the SOCS1 mRNA 3 0 UTR (Figure 5C).Previous studies have demonstrated that miR-19a/b targets the gene SOCS1. 26The target genes of miR-19a/b were subjected to GO and pathway enrichment analysis.A total of 300 target genes were analyzed using the WebGestalt online tool, which yielded a set of biological processes associated with these target genes.The biological processes encompass various categories such as endogenous stimulus responses, cell growth regulation, proliferation, and metabolic processes (Figure S6A).We verified the correlation between IAV replication and SOCS1 expression (Figure 5E).The infection of BJ05/H1N1 in the expression of SOCS1 protein in a manner that was dependent on time.
SOCS1 expression was assessed at various time intervals during BJ05/ H1N1 infection in A549 cells.SOCS1 expression was found to be upregulated in a time-dependent manner in A549 cells infected with BJ05/H1N1, particularly in the early stages of infection (Figure 5F).The level of SOCS1 was detected in A549 cells infected with BJ05/ H1N1 at varying MOIs.The study found that the expression of SOCS1 increased in a dose-dependent manner in A549 cells infected with BJ05/H1N1 (Figure 5G).SOCS1 protein expression was observed in the lungs of three groups of mice.Following agomir-miR-19a/b treatment, ApoE À/À mice exhibited inhibited SOCS1 expression (Figure 5D).Transfection efficiency is shown in Figure S9D.

miR-19a/b enhances JAK/STAT signaling pathway activation
We used lung tissues of BJ05/H1N1 virus mice infected for 5 days, including ApoE À/À +agomir-NC, ApoE À/À +agomir-miR19a/b, and NC, for transcriptome sequencing.By analyzing the three KEGG pathways of ApoE À/À +agomir-NC/NC, ApoE À/À +agomir-miR19a/ b/NC, and ApoE À/À +agomir-miR19a/b/ApoE À/À +agomir-NC, it was found that differential genes were enriched in the JAK/STAT pathway (Figure 6A).It has been demonstrated that NS and M proteins exert a negative regulatory effect on JAK/STAT signal transduc-tion by upregulating the expression of SOCS1 and SOCS3. 27Therefore, we propose that miR-19a/b may upregulate JAK/STAT signal transduction by suppressing SOCS1 expression, leading to inhibition  The activation of the STAT1 signaling cascade during IAV infection was examined by analyzing the phosphorylation of STAT1 at tyrosine 701.This site is crucial for full activation of STAT1 and is specifically targeted in a manner that is dependent on STAT1.Phosphorylation of STAT1 was observed following infection of A549 cells with BJ05/H1N1 virus at an MOI of 1. Increasing the concentration of agomir-miR-19a/b resulted in a greater inhibition of SOCS and an increase in the expression of JAK and p-STAT1.In order to verify that miR-19a/b mainly inhibits viruses by targeting SOCS1, we first constructed the SOCS1knockout (KO) cells and, through re-expression of SOCS1 WT in SOCS1-KO cells, constructed the exogenous SOCS1 overexpression cell line.The two groups were transfected with SOCS1 WT + miR-19a/b and SOCS1 WT +miR-NC, respectively, and then infected with H1N1 virus at MOI = 1 for 24 h, and we detected their protein expression levels to compare the differences.The results showed that the NP levels did not downregulate in exogenous SOCS1 cells, indicating that miR-19a/b mainly targeted SOCS1 to inhibit viral replication.
Inflammatory cytokine expression was assessed at various time intervals, revealing lower levels in the simulant treatment group compared to the CHD group (Figure 6E).The findings suggest that miR-19a/b can activate the JAK/STAT pathway by targeting SOCS1.

SOCS1 promotes influenza virus replication
To further determine the role of SOCS1 in IAV infection, pcDNA3.1-SOCS1,lenti-crisprv2-SOCS1, and empty vector were transfected into A549 cells and overexpressed, and KO SOCS1 cell lines were constructed, which were infected with BJ05/H1N1.As shown in Figure 7A, overexpression of SOCS1 significantly promoted the replication of BJ05/H1N1 and reached a peak value at 36 h, whereas SOCS1 deletion inhibited the expression of NP (Figure 7B), which was consistent with the results of western blot (Figures 7C and 7D).We use one more influenza virus (A/environment/Qinghai/1/2008/ H5N1) strain to verify the major conclusion.A549 cells were transfected with lenti-crisprv2-SOCS1 or pcDNA3.1-SOCS1for 48 h and then infected with H5N1 virus.At 24 and 48 hpi, cells were washed, and the protein was extracted and detected by western blot (Figure S7).RT-qPCR was used to evaluate the expression of inflammatory cytokines at different time intervals, showing that SOCS1 KO could significantly inhibit the expression of inflammatory cytokines (Figures 7E  and 7F).These results clearly identify the role of SOCS1 as a viral factor in the cellular response to BJ05/H1N1 infection.

DISCUSSION
Individuals with CHD are at heightened risk for IAV infection.miR-19a and miR-19b expression is downregulated in patients with CHD compared to healthy individuals.Studies have demonstrated that reduced expression of miR-19a and miR-19b can result in heightened susceptibility to IAV infection, both in vivo and in vitro.Our study indicates that reduced levels of miR-19a and miR-19b may exacerbate the incidence and fatality of influenza virus infection.This offers a novel perspective on comprehending the populace's inclination toward influenza.
The miR-17-92 gene cluster is conserved and has been linked to the development of multiple organ dysfunction syndrome and tumorigenesis in mammals.The six members play significant roles in various settings such as normal development, immune disease, cardiovascular disease, neurodegenerative disease, and aging, among others. 28Recent studies have established the crucial involvement of miRNAs in the innate immune response against viruses.Shimakami et al. 29 found that miR-122 can directly impact the RNA genome of Hepatitis C virus (HCV) during infection, leading to changes in genome stability and increased viral replication.Conversely, certain research groups have yielded contradictory outcomes.Lanford et al. 30 demonstrated that miR-122 can impede the replication of HCV by inducing the degradation of its RNA genome.hsa-miR-122, hsa-miR-199a, hsa-miR-448, hsa-miR-196, and hsa-let-7b have been identified as direct inhibitors of HCV replication.miR-let-7c inhibits the expression of H1N1 influenza virus M1 protein and thereby affects the replication of influenza virus in human lung epithelial cells. 31To date, limited research has explored the possible involvement of the miR-17-92 cluster in the antiviral mechanism.We investigated the mechanism underlying the inhibitory effect of miR-19a and miR-19b on virus infection.SOCS1 was identified as a target of miR-19a and miR-19b in the present study.The SOCS family plays a significant role in regulating the innate immune response triggered by microbial pathogens.Their main role is to regulate negative feedback of cytokine signal transduction through JAK/STAT and other signal pathways. 18,32][35] Certain miRNAs, both viral and host derived, can regulate viral replication by targeting either the viral genome or host genes.This has been documented in previous studies. 36,37During infectious bursal disease (IBD) infection, miR-155 in the host can hinder the expression of SOCS1 and TANK, which ultimately restrains IBDV replication by promoting IFN-I-mediated antiviral response. 38TGEV infection induces endoplasmic reticulum stress and upregulates IRE1a expression, leading to a decrease in host miR-30a-5p levels.This reduction in miR-30a-5p inhibits antiviral responses by decreasing SOCS1 and SOCS3 expression, thereby promoting TGEV replication. 39e precise function of miR19a/b in influenza remains uncertain.This study presents evidence that miR19a/b, through targeting SOCS1, plays a role in mediating influenza-induced cytokine storms both in vitro and in vivo.Subsequent studies have verified that latestage influenza virus-infected cells can increase miR19a/b expression and suppress SOCS1 expression, thus stimulating the JAK/STAT signaling pathway and augmenting the antiviral properties of IFN.Overexpression of SOCS1 reduced phosphorylation of JAK1 and STAT1, leading to inhibition of IFN-I-induced antiviral and antiproliferative responses.The expression of miR19a/b mRNA was found to be suppressed during the initial stages of viral replication (as early as 12 h) in individuals infected with influenza.This observation suggests that the virus may have developed mechanisms to evade the immune system in order to facilitate its replication.
Comprehensive clinical research suggests that influenza virus susceptibility in CHD may be attributed to several mechanisms.Firstly, viral infection can act as an inflammatory stimulus, impacting the body's coagulation function and leading to a decline in antithrombin-III function, thereby creating favorable conditions for the formation of coronary atherosclerotic plaques.Secondly, viral infection can affect the formation of blood lipids, resulting in an increase in the level of low-density lipoprotein and the occurrence of atherosclerotic plaques.Lastly, the body's response to stress after virus infection, combined with the virus itself acting as an inflammatory stimulus, can cause the instability and rupture of atherosclerotic plaques, inducing the expression of IL-8, TNF-a, and other factors.The activation of other cytokines results in the promotion of a heightened inflammatory response, which in turn triggers the rupture of atherosclerotic plaques and subsequently leads to an acute CHD episode.The expression level of miR-19a/b is observed to be downregulated in cases of CHD, accompanied by an increase in the expression level of SOCS1.The protein SOCS1 functions as an inhibitor of the JAK/STAT pathway, thereby promoting inhibition of influenza virus replication.Overall, the presence of miR-19ab as a biomarker in patients with CHD has been linked to an increased susceptibility to influenza virus.Additionally, miR-19a/b has been identified as a potential target for the treatment of influenza and CHD.

Conclusion
Patients with CHD are associated with downregulation of miR-19a/b, and this dysregulation may facilitate IAV infection.Overexpression of miR-19a/b reduces the mortality and prolongs the survival of CHD mice infected with H1N1.miR-19a/b can be considered as a preventative/therapy strategy for patients with CHD against influenza virus infection (Figure S8).

Ethics statement and biosafety
All the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and the protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Zoology, Chinese Academy of Sciences (approval number: IOZ-IACUC-2022-251).All experiments involving influenza viruses were performed in an Animal Biosafety Level 3 containment laboratory in the Research Center for Wildlife Diseases, which was approved by the Chinese Academy of Sciences.

Cells and viruses
A549 cells (human airway epithelial cell line) and MDCK cells (Madin-Darby canine kidney cell line) were purchased from ATCC and cultured in DMEM (Dulbecco's modified Eagle medium; Gibco, London, UK) with 10% fetal bovine serum (Gibco) at 37 C in 5% CO 2 .IAV BJ05/H1N1 (A/Beijing/05/2009(H1N1)) viruses were isolated and stored at the Institute of Zoology, Chinese Academy of Sciences.These viruses were propagated in 10-day-old specific pathogen-free (SPF) chicken embryos (Vital River Laboratories, Beijing, China).Virus titers determined for infection were calculated by plaque assay or 50% tissue culture infectious dose (TCID 50 ) titration on MDCK cells.

Mice
ApoE À/À mice (males, $8 weeks old, $22 g bodyweight) with C57BL/ 6J background were purchased from Beijing SPF Laboratory Animal Technology.The animals were housed in SPF units of the Animal Center at Chinese Academy of Sciences, at 23 C ± 1 C, with a relative humidity of 60%-70% and a 12-h light/dark cycle.The animals can freely access to water and high-fat diet containing 41% fat plus 0.5% cholesterol (MD12015A, Medicience, Jiangsu, China) during the treatment.The animals were checked daily for food and water intake and body weight gain during the treatment.Studies were carried out under animal care guidelines of the Institute of Microbiology, Chinese Academy of Science, with license permit number SCXK20190010.
Isolation of PBMC and total RNA was extracted from the cells Whole blood was absorbed and thoroughly mixed with an equal amount of phosphate-buffered saline (PBS), 2 mL human lymphocyte separation solution was added, and the mixture was centrifuged at 2,000 rpm for 15min.PBMCs were absorbed and placed in 1.5 mL EP tube without enzyme; an appropriate amount of PBS was added and thoroughly mixed, then centrifuged; and precipitate was retained and stored at À80 C for testing.The protocol was approved by the Committee on the Ethics of Experiments of the Institute of Zoology, Chinese Academy of Sciences(No.2016PHB100-01), and all patients gave informed consent.
Normal saline was mixed with agomir-miR-19a, agomir-miR-19b, and agomir-NC.The mice that successfully established atherosclerosis models were randomly divided into an ApoE À/À mice group, ApoE À/À +agomir-miR-19a/b-treated mice, and an agomir-NC mice group, with 10 mice in each group.The ApoE À/À +agomir-miR-19a/ b-treated mice group was injected with a mixture of 5 nmol agomir-miR-19a/b through the tail vein, the NC mice group was injected with a mixture of 5 nmol agomir-NC through the tail vein, and the ApoE À/À mice group was injected with the same amount of agomir-NC through the tail vein every 3 days for 3 weeks.RT-qPCR was used to detect whether miR-19a and miR-19b were overexpressed in ApoE À/À +agomir-miR-19a/b-treated mice.

miRNA quantification
Total RNA was isolated from infected and non-infected cells at different time points using Trizol Reagent (Thermo Fisher Scientific).Total RNA was treated with RNase-free DNase I (Roche, Indianapolis, IN, USA).Then, the total RNA was treated with poly(A) polymerase at 37 C for 1 h in a 20-mL-volume reaction mixture following the manufacturer's instruction.1 mg total RNA was used to synthesize cDNA with a poly(T) adaptor.For each miRNA quantification, 50 ng cDNA was used in a volume of 25 mL mixture.The program was performed as follows: 95 C for 10 min, followed by 40 cycles of 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s.The reactions were run on ABI7500 Fast (Applied Biosystems, Waltham, MA, USA) Virus infection A549 cells were inoculated with BJ05/H1N1 virus at indicated titers in serum-free DMEM for 1 h at 37 C.The cells were washed three times with PBS and then cultured in 0.2 mL influenza virus growth medium, consisting of DMEM supplemented with 1% penicillin and streptomycin antibiotics, 0.2% bovine serum albumin (AMRESCO, Solon, OH, USA), 25 mM HEPES (Life Technologies, Carlsbad, CA, USA), and 1 mg/mL TPCK-treated trypsin (Sigma-Aldrich, St. Louis, MO, USA) at 37 C in 5% CO 2 .

Mouse infection
C57BL/6J mice, ApoE À/À mice, and ApoE À/À +agomir-miR-19a/btreated mice of the same age were challenged by intranasally inoculation.Briefly, mice were lightly anesthetized with CO 2 and intranasally inoculated with PBS and 10 5 TCID 50 BJ/H1N1 influenza virus in a 100 mL volume.Body weight and clinical signs were recorded daily.

IAV quantification
Virus titrations were performed by TCID 50 assays.In brief, cell supernatants or grinding fluid of lung tissues were collected at indicated time points.One day prior to infection, MDCK cells were seeded in 96-well dishes with 3 Â 10 4 cells in 0.2 mL DMEM plus 10% fetal bovine serum and 1% penicillin and streptomycin antibiotics.Confluent monolayer MDCK cells were inoculated with 1 / 2 log 10 serial dilutions of samples using cell media plus TPCK trypsin for dilutions.Cells were washed three times with PBS 1 h after being inoculated and cultured in 0.2 mL influenza virus growth medium.Three days after inoculation, cells were observed for endpoints in cytopathic effect or agglutination activity by using 1% (vol/vol) chicken red blood cells as an 10 indicator of virus replication in the cells.Virus titration was calculated by using the Reed-Muench method.

Pathological examination
Lungs were collected and fixed in 4% paraformaldehyde.Lung tissues were embedded in paraffin and cut into 5-mm sections.The sections were stained with H&E.

Immunofluorescence tissue staining
Lungs were collected and fixed in 4% paraformaldehyde.Lung tissues were embedded in paraffin and cut into 5-mm sections.Briefly, the sections were deparaffinized with serial ethanol and dealt with antigen retrieval buffer preceding permeabilization with 0.5% Trition-100.Virus antigen was stained using a monoclonal antibody of H1N1 influenza virus NP protein and visualized with a secondary goat anti-rabbit antibody conjugated to fluorescein isothiocyanate.Sections were also stained for DNA with DAPI (Sigma).

Cytokine or chemokine analysis
Lungs were collected and homogenized on days 1, 2, 3, 5, and 7 postinfection.Total RNA samples were extracted using Trizol Reagent.Briefly, TNF-a, IFN-g, IL-1b, IL-4, IL-6, and MIP-1a were quantified using SYBR I-based real-time PCR.The results were normalized to GAPDH and WT mice on day 0 post-infection.The primers used in this assay are listed in Figure S10.

Western blot
The proteins were extracted from the lung tissues using RIPA lysis (Beyotime, Shanghai, China) and then measured by BCA kit (Beyotime) to determine protein concentration.After that, the proteins were mixed with loading buffer in a boiled water bath for 3 min for dena-ture.Then, the proteins were subjected to electrophoresis at 80 V for 30 min and then 120 V for 1-2 h.The proteins were transferred into the membrane on ice bath at 300 mA for 60 min.The membranes were blocked in confining liquid for 60 min or 4 C overnight before being incubated into primary antibody for b-actin (44035, 1:1,000), NP (125989, 1:1,000), M1 (127356, 1:1,000), SOCS1 (37038, 1:1,000), JAK1 (bs-1439R, 1:1,000), pSTAT1 (ab30645, 1:1,000), and STAT1 (ab31369, 1:1,000) (all from Cell Signaling, Boston, MA, USA) for 1 h.After incubation, the membranes were washed in washing buffer for 3 Â 10 min before secondary antibody was applied for incubation at room temperature for 1 h.After being washed for 3 Â 10 min, the membranes were added with developer solution for color development.The color development was verified in a chemiluminescent imaging system (Bio-Rad, Hercules, CA, USA).

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0.2.Statistical significance was determined by two-tailed unpaired Student's t test or by one-way/two-way ANOVA, and p values <0.05 are considered significant and denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.Non-significant values are denoted as ns.

Figure 1 .
Figure 1.Expression of miR-19a/miR-19b in the coronary heart disease of patients and mice (A) The basic information of patients with coronary heart disease and healthy people.(B) RT-qPCR of miR-19a and miR-19b in patients with coronary heart disease in PBMCs.n = 15.(C) Schematic diagram of experimental protocol for coronary heart disease mouse model.(D) Myocardial contractility (FS, fractional shortening).(E) Biochemical indicators of NC mice and ApoE À/À mice.(F) ApoE À/À mice and healthy mice of miRNA sequencing in lungs.(G) Scheme of miRNA quantification process.(H) KEGG enrichment of miRNA target genes in lungs of ApoE À/À mice and NC mice.Data are presented as mean ± SD.Asterisks denote the significance levels: *p < 0.05, **p < 0.01, and ***p < 0.001.
of viral infection.To examine the impact of miR-19a/b on SOCS1 protein levels, A549 cells were both infected with BJ05/H1N1 (MOI = 1) and transfected with varying concentrations of agomir-miR-19a/b.Transfecting agomir-miR-19a/b into A549 cells resulted in a significant decrease in the expression of SOCS1 and influenza protein.miR-19a/b has demonstrated a dose-dependent suppression of SOCS1 protein, and there was no change in the agomir-NC group (Figure 6B).