Integrated analysis of microRNA-mRNA expression in A549 cells infected with influenza A viruses (IAVs) from different host species
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.
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The first two authors contributed equally to the work.