Elsevier

Virus Research

Volume 263, 2 April 2019, Pages 34-46
Virus Research

Integrated analysis of microRNA-mRNA expression in A549 cells infected with influenza A viruses (IAVs) from different host species

https://doi.org/10.1016/j.virusres.2018.12.016Get rights and content

Highlights

  • It is the first integrated analysis of miRNA-mRNA expression in A549 cells infected with IAVs from different host species.

  • Twenty miRNAs and 1286 mRNAs were differentially expressed in all three infected groups.

  • A GO enrichment analysis showed that 107 miRNA-mRNA interactions were correlated with the defense of the virus.

  • A KEGG enrichment analysis showed that 79 miRNA-mRNA interactions were involved in the influenza A reference pathway.

Abstract

Although several miRNAs have been demonstrated to be involved in the influenza virus replication cycle, the identification of miRNAs and mRNAs that are expressed in A549 cells infected with influenza A viruses (IAVs) from different host species has remained poorly studied. To investigate the molecular mechanisms associated with the differential expression of miRNAs during influenza A virus infection, we performed global miRNA and mRNA expression profiling in A549 cells infected with human-origin seasonal influenza A virus H3N2 (Human_Br07), swine-origin influenza A virus H1N1 (SW_3861) or avian-origin influenza A virus H3N2 (AVI_9990). The miRNA and mRNA expression profiles were obtained by microarray and high-throughput sequencing analyses, respectively. The integrated analysis of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was performed using bioinformatics tools, and the expression of miRNAs and mRNAs was validated by real-time quantitative polymerase chain reaction (RT-qPCR). We identified 20 miRNAs (6 upregulated and 14 downregulated) and 1286 mRNAs (935 upregulated and 351 downregulated) exhibiting the same differential expression trends in three infected groups of cells compared with an uninfected control. An integrated analysis of these expression profiles identified 79 miRNA-mRNA pairs associated with the influenza A reference pathway, and 107 miRNA-mRNA interactions were correlated with the defense of the virus. Additionally, the obtained results were supported by an RT-qPCR analysis of 8 differentially expressed miRNAs (hsa-miR-210-3p, hsa-miR-296-5p, hsa-miR-371a-5p, hsa-miR-762, hsa-miR-937-5p, hsa-miR-1915-3p, hsa-miR-3665, and hsa-miR-1290) and 13 differentially expressed mRNAs (IFNL1, CXCL10, RSAD2, MX1, OAS2, IFIT2, IFI44 L, MX2, XAF1, NDRG1, FGA, EGLN3, and TFRC). Our findings indicate that dysregulated miRNA expression plays a crucial role in infection caused by IAVs originating from different species and provide a foundation for further investigations of the molecular regulatory mechanisms of miRNAs involved in influenza A virus infection.

Introduction

Influenza A virus (IAV) is an enveloped negative-sense single-stranded RNA virus belonging to the Orthomyxoviridae family. Individuals infected with this virus exhibit several respiratory symptoms, including fever, cough, headache, fatigue, runny nose and sore throat, and in some cases such infections can be fatal (Nicholson et al., 2003), especially in instances of interspecies transmission (Short et al., 2015). Generally, IAV exhibits species specificity, but viruses from other species, including avian and swine viruses, can also infect humans by crossing the species barrier (Kuiken et al., 2006; Van Reeth, 2007). In recent years, IAVs of avian- and swine-origin have caused death in several cases and present a severe threat to human health (Sun et al., 2018; Zhou et al., 2018). Besides, IAVs of human-origin cannot be ignored, as the seasonal virus may cause 290,000–650,000 deaths each year (http://www.who.int/news-room/detail/14-12-2017-up-to-650-000-people-die-of-respiratory-diseases-linked-to-seasonal-flu-each-year, Accessed 9 January 2019) and has resulted in serious physical and economic burdens on the human population. Although the pathogenic mechanisms of IAVs have continued to be studied, few investigations have simultaneously studied the pathogenicity of IAVs from different species. In this study, three IAVs from different species were used to infect human lung carcinoma cells (A549) to study common aspects of their pathogenicity.

MicroRNAs (miRNAs) are endogenous, small (17–24 nucleotides in length) noncoding RNAs that have been identified in animals, plants and some viruses (Bartel, 2004; Carthew and Sontheimer, 2009; Grundhoff and Sullivan, 2011), regulating target gene expression at the posttranscriptional level via mRNA degradation or translational repression (Turchinovich et al., 2012). As of March of 2018, 48,885 human mature miRNAs were registered in the miRBase 22 release, and the number of newly discovered miRNAs is still growing. For influenza virus, only one study has reported on an IAV-encoded microRNA-like small RNA (miR-HA-3p), which is encoded by an H5N1 virus (Li et al., 2018), with most miRNAs involved in influenza virus infection being produced by host cells. Previous studies showed that miRNAs have key regulatory roles in cellular biological processes, including cell proliferation, differentiation, metabolism and apoptosis (Bartel, 2004; Zhang et al., 2018b) as well as in regulating disease development, such as cancer formation, and pathogen infection (Hill and Tran, 2018; Holla and Balaji, 2015; Trobaugh and Klimstra, 2017). Recently, many studies have reported that the expression profiles of host miRNAs are altered upon infection by IAVs, demonstrating that some miRNAs may participate in the influenza virus infection process. For example, let-7c, miR-323, miR-491, miR-654, miR-3145, miR-584-5p and miR-1249 have been reported to target the viral genes M1, PB1 or PB2 to directly inhibit viral replication (Khongnomnan et al., 2015; Ma et al., 2012; Song et al., 2010; Wang et al., 2017). In addition, miR-21-3p, miR-144, miR-146a, miR-203, miR-302 and miR-483-3p were shown to target host genes to indirectly mediate the antiviral response by inducing the generation of immune factors (Chen et al., 2017; Deng et al., 2017; Maemura et al., 2018; Rosenberger et al., 2017; Xia et al., 2018; Zhang et al., 2018a). In 2012, Emma-Kate Loveday et al. provided the first experimental evidence demonstrating the complex temporal and strain-specific regulation of the host microRNAome by pandemic S-OIV and deadly A-OIV-host infections in human cells (Loveday et al., 2012). Subsequently, Jarika Makkoch et al. investigated the miRNA expression profiles of A549 cells infected with different influenza virus subtypes (pH1N1, H3N2 and H5N1) (Makkoch et al., 2016). However, to date, a comprehensive analysis of miRNAs and mRNAs that are commonly expressed in A549 cells infected with different typical influenza viruses from a variety of species sources has not been reported.

In this study, we performed global miRNA and mRNA expression profiling in A549 cells infected with three types of IAVs from different host species (human, swine and avian origin) and explore the molecular regulatory pathways of miRNAs with common expression patterns among the three IAVs infection, which may be involved in the IAV infection process. In addition, real-time quantitative polymerase chain reaction (RT-qPCR) was used to test whether the expression of miRNAs and mRNAs was consistent with the results of the microarray and high-throughput sequencing analyses. The findings of this study provide novel insights into anti-IAV mechanisms and may be helpful in guiding research aimed at delineating broad-spectrum antiviral targets for pandemic influenza control.

Section snippets

Cell culture and virus infection

The human lung carcinoma cells (A549) used in this study were cultured in Kaighn’s modification of Ham’s F-12 K Medium (F12 K; Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin in a 37℃ incubator with 5% CO2. The influenza viruses A/Brisbane/10/2007(H3N2), A/Duck/Shantou/9990/2010(H3N2) and A/Swine/Guangxi/3861/2011(H1N1) were propagated in specific pathogen-free (SPF) embryonated eggs. The harvested viruses were stored at −80℃ prior to use. All viruses were titrated on

miRNA expression profiling

To identify changes in miRNA expression in A549 cells infected with influenza A virus, four groups were assayed, including the Human_Br07 H3N2-infected group, the AVI_9990 H3N2-infected group, the SW_3861 H1N1-infected group and an uninfected group. Each group was assayed in triplicate, and 12 small RNA libraries were constructed for miRNA expression profiling. Agilent Human miRNA Microarrays (Release 21.0) provided 2549 human miRNA probes. The DEMs of the three infected groups were screened

Discussion

IAV infection is a constant threat to humans, and many studies have indicated that miRNAs play key roles in the IAV infection process (Buggele et al., 2012; Peng et al., 2018). However, there is a lack of research on miRNAs involved in infections by IAVs originating from different species, and few integrated analyses of DEMs and DEGs during IAV infections have been performed. In our study, we identified 20 miRNAs and 1286 mRNAs that are differentially expressed in A549 cells infected by all

Authors’ contributions

JG, LXG, RL, ZPL and ZFZ carried out the experiments, JG and LXG analyzed and synthesized the data and wrote the manuscript. XHF designed the study and revised the manuscript. All authors read and approved the final manuscript.

Conflicts of interest

The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Acknowledgements

This study has been supported by National Natural Science Foundation of China (No. 31660040), Innovating Project of Guangxi Graduate Education and YCBZ2014027 from Guangxi Education Department, Young Scientist Foundation of Guangxi Medical University (Grant No. GXMUYSF201525), and Promotion Ability Project of Young Teachers in Guangxi Universities (Grant No. 2018KY0138). The funders had no role in the study design, data collection and analysis, or preparation of the manuscript.

References (51)

  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods

    (2001)
  • K.G. Nicholson et al.

    Influenza

    Lancet

    (2003)
  • F. Peng et al.

    Identification of serum MicroRNAs as diagnostic biomarkers for influenza H7N9 infection

    Virol. Rep.

    (2017)
  • S. Peng et al.

    Endogenous Cellular microRNAs mediate antiviral defense against influenza A virus. Molecular therapy

    Nucleic Acids

    (2018)
  • K.R. Short et al.

    One health, multiple challenges: The inter-species transmission of influenza A virus

    One Health

    (2015)
  • D.W. Trobaugh et al.

    MicroRNA regulation of RNA virus replication and pathogenesis

    Trends Mol. Med.

    (2017)
  • A. Turchinovich et al.

    Extracellular miRNAs: the mystery of their origin and function

    Trends Biochem. Sci.

    (2012)
  • G.L. Zhang et al.

    Suppression of hepatitis B virus replication by microRNA-199a-3p and microRNA-210

    Antiviral Res.

    (2010)
  • S. Anders et al.

    HTSeq--a Python framework to work with high-throughput sequencing data

    Bioinformatics

    (2015)
  • Simon Anders et al.

    Differential Expression of RNA-Seq Data at the Gene Level –The DESeq Package

    (2012)
  • S. Holla et al.

    Epigenetics and miRNA during bacteria-induced host immune responses

    Epigenomics

    (2015)
  • J.J. Jing et al.

    Key elements involved in Epstein-Barr virus-associated gastric cancer and their network regulation

    Cancer Cell Int.

    (2018)
  • M. Kanehisa et al.

    KEGG for linking genomes to life and the environment

    Nucleic Acids Res.

    (2008)
  • Y. Kawano et al.

    Analysis of circulating human and viral microRNAs in patients with congenital cytomegalovirus infection

    J. Perinatol.

    (2016)
  • K. Khongnomnan et al.

    Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene

    Exp. Biol. Med.

    (2015)
  • Cited by (30)

    • Exosomal mediated signal transduction through artificial microRNA (amiRNA): A potential target for inhibition of SARS-CoV-2

      2022, Cellular Signalling
      Citation Excerpt :

      It induces RNA degradation or translation [48,49]. The miRNA between MRE and base sequence of six to eight 5′ end of matured miRNA known to be seed miRNA [50] This miRNA-mRNA is correlated with antiviral mechanism in cells [55]. The predicted miRNA is to be better understood of Cov-2- SARS infection.

    • Exosomal microRNA expression profiles of cerebrospinal fluid in febrile seizure patients

      2020, Seizure
      Citation Excerpt :

      Additionally, several studies revealed the relationship between infection and miRNAs [37,38]. One such study analyzed miRNA-mRNA expression in A549 cells (influenza A virus infected) [39]. On the other hand, a study reported that miR-671-5p, miR-16-5p, miR-150-3p, and miR-4281 levels differed significantly in exosomes of patients with hand, foot, and mouth disease when compared to those of the control [24].

    • Identification of a novel antiviral micro-RNA targeting the NS1 protein of the H1N1 pandemic human influenza virus and a corresponding viral escape mutation

      2019, Antiviral Research
      Citation Excerpt :

      Among them 15 (50%) regulate cell proliferation and apoptosis, mostly in a positive fashion, 7 (23%) were involved in the regulation of the inflammatory and IFN response, while the remaining 8 (27%) were involved in the regulation of various metabolic pathways. Hundreds of cellular miRNAs are differentially expressed upon IAV infection (Ma et al., 2016; Gao et al., 2019) and some also specifically target IAV proteins, such as NP, PB1, PB2, PA, and NA (Nguyen et al., 2018; Kumar et al., 2018; Gao et al., 2019; Jiao et al., 2019). However, to the best of our knowledge, no human miRNA has been validated so far as a suppressor of the IAV NS1 protein expression.

    View all citing articles on Scopus
    1

    The first two authors contributed equally to the work.

    View full text