上海交通大学学报(医学版) ›› 2024, Vol. 44 ›› Issue (3): 301-311.doi: 10.3969/j.issn.1674-8115.2024.03.002
• 论著 · 基础研究 • 上一篇
徐文晖(), 杨畅, 李瑞卿, 卞京, 李夏伊, 郑磊贞()
收稿日期:
2023-05-14
接受日期:
2024-03-19
出版日期:
2024-03-28
发布日期:
2024-04-29
通讯作者:
郑磊贞
E-mail:xuwenhui98@126.com;zhengleizhen@xinhuamed.com.cn
作者简介:
徐文晖(1998—),女,硕士生;电子信箱:xuwenhui98@126.com。
基金资助:
XU Wenhui(), YANG Chang, LI Ruiqing, BIAN Jing, LI Xiayi, ZHENG Leizhen()
Received:
2023-05-14
Accepted:
2024-03-19
Online:
2024-03-28
Published:
2024-04-29
Contact:
ZHENG Leizhen
E-mail:xuwenhui98@126.com;zhengleizhen@xinhuamed.com.cn
Supported by:
摘要:
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。
中图分类号:
徐文晖, 杨畅, 李瑞卿, 卞京, 李夏伊, 郑磊贞. 干扰素调节因子3促结直肠癌细胞增殖与侵袭相关探索[J]. 上海交通大学学报(医学版), 2024, 44(3): 301-311.
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.
图1 基于不同IRF3表达水平的Kaplan-Meier生存分析
Fig 1 Kaplan-Meier survival analysis based on different IRF3 expression levelsNote: A. Renal cancer. B. Colorectal cancer. C. Liver cancer. D. Prostate cancer.
图2 肠癌患者癌组织与癌旁正常组织IRF3蛋白免疫组织化学对比(×20)
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.
图3 肾癌患者癌组织与癌旁正常组织的IRF3免疫组织化学对比(×20)
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.
图5 3组间差异基因聚类热图Note: Horizontal coordinates are experimental groups. A1: IRF3-WT48 h, A2: IRF3-WT72 h; B1: IRF3-5D48 h, B2: IRF3-5D72 h; C1: IRF3-2A5D48 h, C2: IRF3-2A5D72 h. Vertical coordinates are the names of the major differential genes detected at the transcriptional level. Blue via white to red indicates low to high expression; red indicates high expression genes, and blue indicates low expression genes. CDRT4—CMT1A duplicated region transcript 4; SLFN5—recombinant Schlafen family member 5; RASA4—RAS p21 protein activator 4; MALAT-1—metastasis associated in lung denocarcinoma transcript 1; TOMM6—translocase of outer mitochondrial membrane 6; BISPR—BST2 interferon-stimulated positive regulator; IFITM3—interferon-induced transmembrane protein 3; CCN3—cellular communication network factor 3; B2M—β-2 microglobulin; HELZ2—helicase with zinc finger 2; IRF9—interferon regulatory factor 9; IRF7—interferon regulatory factor 7; DDX60—DExD/H-box helicase 60; PARP9—poly (ADP-ribose) polymerase family member 9; SP110—SP110 nuclear body protein; OAS2—2′-5′-oligoadenylate synthetase 2; CMPK2—cytidine monophosphate kinase 2; RSAD2—radical S-adenosyl methionine domain containing 2; IFI27—interferon alpha-inducible protein 27; BST-2—bone marrow stromal antigen 2; PLSCR1—phospholipid scramblase 1; EPSTI1—epithelial stromal interaction 1; PARP10—poly (ADP-ribose) polymerase family member 10; REC8—REC8 meiotic recombination protein; TRIM22—tripartite motif containing 22; PNPT1—polyribonucleotide nucleotidyltransferase 1; BTK—Bruton tyrosine kinase; STAT2—signal transducer and activator of transcription 2; DTX3L—deltex E3 ubiquitin ligase 3L; HERC6—HECT and RLD domain containing E3 ubiquitin protein ligase family member 6; XAF1—XIAP-associated factor 1; DHX58—DExH-box helicase 58; APOL6—apolipoprotein L6; PRORP—protein-only RNase P catalytic subunit; CCN4—cellular communication network factor 4; SAMD9L—sterile α motif domain containing 9 like; TRIM21—tripartite-motif protein 21; DHX58—DExH-box helicase 58; ZNFX1—zinc finger NFX1-type containing 1; MATR3—matrin 3.
Fig 5 Heat map of differential gene clusterings between the three groups
图8 3组CT26、COLON26细胞增殖能力对比Note: ①P=0.000, compared with CT26Vector; ②P=0.000, compared with COLON26Vector.
Fig 8 Comparison of growing capacity among three groups of CT26 and COLON26 cell
图9 3组CT26细胞转移侵袭能力对比Note: A. Detection of migration capacity of CT26 cells. B. Statistical analysis of CT26 cell wound closure percentage. ①P=0.000, compared between IRF3-5D andVector; ②P=0.000, compared between IRF3-5D and IRF3-WT.
Fig 9 Comparison of invasive ability among three groups of CT26 cells
图10 3组COLON26细胞生长活力对比Note: Initial cell number laid down was 500 and 2 000. A. Colony formation of COLON26 cells. B. Statistical analysis of clone formation rate of COLON26 cells. ①P=0.000, compared between IRF3-5D and Vector; ②P=0.000, compared between IRF3-5D and IRF3-WT; ③P=0.001, compared between IRF3-5D and Vector; ④P=0.001, compared between IRF3-5D and IRF3-WT.
Fig 10 Comparison of cell viability among three groups of COLON26 cells
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