流感大流行和每年全世界范围内都会出现的季节性流感，给全球卫生系统带来极大的医疗经济负担。甲型流感病毒，即A型流感病毒（influence A virus, IAV），是目前全球范围内造成流感流行的主要病毒。流感病毒极易变异、亚型众多以及宿主广泛，对它的预防至今都是一大难题。为了更好的预防和治疗流感相关疾病，我们需要更深入的了解IAV的转录、复制及其致病机理。

甲型流感病毒感染人体后，体内宿主细胞会产生干扰素(IFNs)。干扰素信号可通过JAK-STAT途径诱导数百种干扰素刺激基因（interferon stimulated genes, ISGs）的表达，进而起到抗病毒感染的作用。干扰素抵御流感病毒属于机体天然免疫即固有免疫系统。大多数ISGs抗流感病毒的机制仍是一片空白。

在流感病毒的研究中，我们根据文献调研和实验室前期研究成果总结，选取了22个感兴趣的ISGs，并通过CRISPR/Cas9基因敲除技术构建了人肺癌上皮细胞系A549的基因敲除细胞库。首先，我们采用噬斑实验检测所选ISGs对流感病毒的抑制作用。用流感病毒感染22株ISGs敲除后的细胞系，并在24和48小时后收取上清并进行噬斑实验测定病毒滴度进行统计。统计结果发现GATA3 敲除后细胞对流感病毒的生成有明显的促进作用。为进一步确定GATA3抗流感病毒功能，我们利用Western Blot、PCR以及基因测序确认GATA3 敲除成功，并用细胞活性试验验证敲除GATA3 后对细胞活性并无影响；我们用干扰素和poly(I:C)刺激野生型和GATA3 敲除后的细胞，确认了GATA3 属于干扰素刺激基因。用GATA3 敲除和过表达细胞系分别感染流感病毒，利用流式实验、噬斑实验、RT-PCR以及Western Blot确认了敲除GATA3 能促进流感病毒的生成，而过表达GATA3 则抑制流感病毒的生成。我们还进行了免疫荧光共聚焦实验和免疫荧光流式实验定性、定量的观察到了GATA3 基因敲除后流感病毒生成明显增加的现象。在抗流感病毒机制研究方面，我们首先做了病毒Binding实验、Entry实验以及Replicon实验，实验结果表明GATA3主要通过抑制流感病毒的复制来发挥抗病毒作用。随后通过免疫共沉淀实验探索GATA3跟流感病毒各蛋白之间是否存在相互作用。实验结果发现GATA3可与PB2相互作用抑制PB2与PB1相结合阻碍RdRp的组装从而发挥抗流感病毒的作用。我们通过截短体实验也发现GA作为目前实验验证过的GATA3 最小截短体，发挥抑制流感病毒复制的作用更强。

GATA3与癌症之间的研究已有相关报道，但GATA3泛癌生信分析还没有报道过。因此，我们的第二部分GATA3泛癌生信分析研究从基因差异性表达、肿瘤患者临床分期、生存预后、基因突变和肿瘤发生、免疫细胞浸润相关性以及基因相关的细胞通路等方面分析了GATA3与不同癌症的临床相关性及其可能的作用机制。

综上，我们的病毒研究确定了GATA3是一种新的抗病毒ISG，并且是通过干扰病毒复制过程增强机体抗病毒免疫应答，为ISG抗病毒机理研究方向提供了新的线索，丰富了对天然免疫抗病毒作用机制的认识，截短体实验研究的新发现更为抗流感病毒药物的研发提供了新的思路。我们的泛癌分析为GATA3在不同肿瘤中的作用机制提供了一个相对全面的生信数据研究成果，为GATA3成为乳腺癌临床分子指标提供了生信大数据支持。

Influenza pandemics and seasonal influenza, which occur annually worldwide, place a significant medical and economic burden on global health systems. Influenza A virus (IAV) is currently the main virus responsible for influenza pandemics worldwide. The highly variable nature of influenza viruses, their many subtypes and the wide range of hosts make their prevention a challenge to date. In order to better prevent and treat influenza-related illnesses, we need to better understand the transcription, replication and pathogenesis of IAV.

Influenza A virus infection in humans results in the production of interferons (IFNs) by host cells in the body. Interferon signal induces the expression of hundreds of interferon stimulated genes (ISGs) through the JAK-STAT pathway, which in turn acts as an antiviral agent. Interferon resistance to influenza viruses is part of the body's natural immunity, the innate immune system. Most of the mechanisms of ISGs against influenza viruses are still unknown.

In this paper, we selected 22 ISGs of interest and constructed a knockout cell library of human lung cancer epithelial cell line A549 by CRISPR/Cas9 knockout technology based on literature research and summary of previous laboratory findings. Firstly, we used phagocytic spot assay to detect the inhibitory effect of the selected ISGs on influenza virus.22 ISGs knockout cell lines were infected with influenza virus and the supernatants were collected after 24 and 48 hours and the virus titers were measured by phagocytosis assay for statistical purposes. The statistical results revealed a significant promotion of influenza virus production by the GATA3 knockout cells. To further determine the anti-influenza function of GATA3, we confirmed the successful knockout of GATA3 using Western Blot, PCR and gene sequencing, and verified that knockout of GATA3 had no effect on cellular activity using cellular activity assays; we stimulated wild-type and GATA3 knockout cells with interferon and poly(I:C) and confirmed that GATA3 belongs to the interferon-stimulated gene. GATA3 knockout and over-expression cell lines were infected with influenza viruses, and we confirmed that knockout of GATA3 promoted influenza virus production and over-expression of GATA3 suppressed influenza virus production using flow assays, phage spot assays, RT-PCR and Western Blot. We also performed immunofluorescence confocal assays and immunofluorescence flow assays to qualitatively and quantitatively observe a significant increase in influenza virus production after GATA3 knockout. In terms of anti-influenza virus mechanism, we first performed viral Binding assays, Entry assays and Replicon assays, which showed that GATA3 exerts antiviral effects mainly by inhibiting the replication of influenza virus. We then investigated the interaction between GATA3 and influenza virus proteins through immunoprecipitation assays. The results showed that GATA3 interacted with PB2 to inhibit the binding of PB2 to PB1 and hinder the assembly of RdRp, thus exerting anti-influenza viral effects. Our truncation experiments also revealed that GA, the smallest truncator of GATA3 so far validated, exerts a stronger effect on influenza virus replication.

Studies on the association between GATA3 and cancer have been reported, but a pan-cancer analysis of GATA3 has not yet been reported. Therefore, we have analyzed the clinical relevance of GATA3 to different cancers and its possible mechanisms of action in terms of differential gene expression, clinical staging of tumour patients, survival prognosis, gene mutation and tumourigenesis, immune cell infiltration relevance and gene-related cellular pathways.

In summary, our viral studies identified GATA3 as a new antiviral ISG and enhanced the body's antiviral immune response by interfering with the viral replication process, providing new clues to the direction of the antiviral mechanism of ISGs, enriching the understanding of the natural immune antiviral mechanism of action, and providing new ideas for the development of anti-influenza viral drugs with the new findings from truncated in vivo experimental studies. Our pan-cancer analysis provides a relatively comprehensive report for the mechanism of action of GATA3 in different tumors, and provides support for GATA3 to become a clinical molecular indicator in breast cancer.