Exploratory study of interferon regulatory factor 3 promoting proliferation and invasion related to colorectal cancer cells

doi:10.3969/j.issn.1674-8115.2024.03.002

Abstract

Abstract:

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.

Key words: colorectal malignant tumour, targeted therapy, interferon regulatory factor 3 (IRF3), TANK-binding kinase 1 (TBK1), RNA sequencing (RNA-seq)

CLC Number:

R735.3⁺5

XU Wenhui, YANG Chang, LI Ruiqing, BIAN Jing, LI Xiayi, ZHENG Leizhen. Exploratory study of interferon regulatory factor 3 promoting proliferation and invasion related to colorectal cancer cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(3): 301-311.

Figures/Tables 10

Fig 1 Kaplan-Meier survival analysis based on different IRF3 expression levelsNote: A. Renal cancer. B. Colorectal cancer. C. Liver cancer. D. Prostate cancer.

Fig 2 Comparison of immunohistochemical staining for IRF3 protein between cancerous and normal tissues adjacent to cancer in patients with colorectal cancer （×20）Note: A. Normal intestinal tissue adjacent to cancer. B. Cancerous tissue.

Fig 3 Comparison of immunohistochemical staining for IRF3 protein between cancerous and normal tissues adjacent to cancer from patients with renal cancer （×20）Note: A. Normal kidney tissue adjacent to cancer. B. Cancerous tissue.

Fig 4 Changes in IRF3 expression with TBK1 inhibitor at different time points

Fig 5 Heat map of differential gene clusterings between the three groups

Fig 6 Gene enrichment analysis graphs of GO（A） and KEGG（B）

Fig 7 Comparison of the results of the four immunoblots

Fig 8 Comparison of growing capacity among three groups of CT26 and COLON26 cell

Fig 9 Comparison of invasive ability among three groups of CT26 cells

Fig 10 Comparison of cell viability among three groups of COLON26 cells

TrendMD

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