目的·分析结直肠癌中干扰素调节因子3（interferon regulatory factor 3，IRF3）表达水平与临床病理特征及患者预后的关系，观察IRF3过表达对结直肠癌细胞增殖与侵袭能力的影响及其相关蛋白通路。方法·下载癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据，分析IRF3表达水平与恶性肿瘤患者（肾癌、结直肠癌、肝癌、前列腺癌）预后的关系。采用免疫组织化学法检测10例结直肠癌或肾癌患者的癌组织与癌旁正常组织切片中IRF3表达水平的差异。针对IRF3蛋白的C端残基位点进行改造，构建拟磷酸化IRF3-5D（396/398/402/404/405-D）高表达的HEK-293T细胞。分别在细胞培养12、24 h时，采用TANK结合激酶1（TANK-binding kinase 1，TBK1）抑制剂进行处理，并采用蛋白质印迹法检测细胞IRF3、p-IRF3（Ser386）蛋白表达水平。采用RNA测序技术探索IRF3-5D高表达与肿瘤相关蛋白表达水平的相关性。构建野生型IRF3（IRF3-WT）和IRF3-5D过表达的结直肠癌细胞CT26、COLON26，采用细胞计数法、细胞划痕试验和克隆形成试验检测细胞增殖及迁移能力。结果·TCGA数据分析提示癌组织中IRF3蛋白表达水平与患者的不良预后呈正相关。癌症患者病理组织免疫组织化学法显示，结直肠癌、肾癌组织中IRF3的表达水平显著上调，且蛋白表达集中于细胞核内。TBK1抑制剂分别在细胞培养12、24 h时间点作用后，HEK-293T细胞p-IRF3（Ser386）蛋白表达减弱。RNA测序和蛋白质印迹法结果显示，多个与癌症预后不良相关的蛋白［IRF9、细胞程序性死亡-配体1（programmed cell death 1-ligand 1，PD-L1）等］表达水平在IRF3-5D高表达的条件下显著上调。结直肠癌细胞中过表达IRF3-5D，可导致癌细胞的增殖、迁移能力显著上调。结论·结直肠癌中IRF3表达水平与患者不良预后呈正相关。IRF3-5D蛋白在结直肠癌细胞内高表达后，促进癌细胞恶性生物学行为。此外，IRF3-5D依赖于TBK1介导的IRF3活化激活通路，并上调多个肿瘤相关蛋白的表达水平。

Objective ·To analyze the relationship between the expression level of interferon regulatory factor 3 (IRF3) in colorectal cancer and its clinicopathological features and prognosis, and to observe the effects of IRF3 overexpression on the proliferation and invasion ability of colorectal cancer cells and the related protein molecular pathways. Methods The Cancer Genome Atlas (TCGA) data were downloaded and used to analyze the correlation between expression levels of IRF3 and the prognosis of patients (including renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, and prostate cancer). Immunohistochemistry was used to detect the differences in the expression levels of IRF3 between cancerous tissue and adjacent normal tissues of 10 patients with colorectal/renal cancer. The C-terminal residue sites of the IRF3 protein were modified to construct HEK-293T cells overexpressing the phosphorylated IRF3-5D (396/398/402/404/405-D). At 12 and 24 h of cell culture, treatment with TANK-binding kinase 1 (TBK1) inhibitor was performed, and Western blotting was used to detect the expression levels of IRF3 and p-IRF3 (Ser386) in the cells. RNA sequencing (RNA-seq) was employed to explore the correlation between high expression of IRF3-5D and the expression levels of tumor-related proteins. Colorectal cancer cells CT26 and COLON26 overexpressing wild-type IRF3 (IRF3-WT) and IRF3-5D were construct, and cell proliferation and migration ability were assessed by using cell counting, scratch assay, and clonogenic assay. Results ·Analysis of TCGA data suggested that the expression level of IRF3 protein in cancer tissues was positively correlated with poor prognosis in patients. Immunohistochemical analysis of pathological tissues from patients with cancer showed that the expression level of IRF3 was significantly upregulated in colorectal cancer tissues and renal cancer tissues, with protein expression concentrated in the cell nucleus. After treatment with TBK1 inhibitors for 12 and 24 h in cell culture, the expression of p-IRF3 (Ser386) protein in HEK-293T cells decreased. The results of RNA-seq and Western blotting showed that the expression levels of multiple proteins associated with poor prognosis [such as IRF9, programmed cell death 1-ligand 1 (PD-L1), etc.] were significantly upregulated under conditions of high expression of IRF3-5D. Overexpression of IRF3-5D in colorectal cancer cells could significantly enhance the proliferation and migration capabilities of cancer cells. Conclusion ·The expression level of IRF3 in colorectal cancer is positively correlated with poor patient prognosis. High expression of IRF3-5D protein in colorectal cancer cells can promote malignant biological behavior of cancer cells. Additionally, IRF3-5D is dependent on the TBK1-mediated activation of the IRF3 activation pathway and upregulates the expression levels of multiple tumor-related proteins.