补体B因子在结直肠癌中的表达及临床意义

doi:10.3877/cma.j.issn.1674-0793.2024.03.004

目的

基于生物信息学与细胞实验探究补体B因子（CFB）在结直肠癌中的表达及临床意义。

方法

利用HPA和TIMER 2.0数据库分析CFB在结直肠癌组织中的表达；GEPIA数据库分析CFB与肿瘤生长因子MKI67在结直肠癌中表达的关联；Kaplan-Meier Plotter数据库分析CFB mRNA的表达水平与结直肠癌患者生存期的关系；基因富集通路预测CFB对结直肠癌潜在的生物学功能；免疫组织化学（IHC）和Western blotting验证CFB在结直肠癌组织中的表达；采用siRNA转染，利用细胞划痕、Transwell小室实验探究CFB对结直肠癌细胞迁移、侵袭的影响。

结果

HPA和TIMER2.0数据库显示CFB在结直肠癌组织中高表达（P<0.001）。GEPIA数据库显示结直肠癌组织中CFB表达较高（P<0.05），且与MKI67表达呈正相关（P=0.004 1）。Kaplan-Meier Plotter生存分析表明，低表达的CFB在结直肠癌中预示更差的总生存期（P=0.015）和进展后生存期（P=0.048）。基因富集结果显示，CFB可能参与多个结直肠癌进展通路。IHC显示，CFB在结直肠癌组织中阳性染色强度高于正常组织，且在TNMⅠ、Ⅱ期中的表达弱于Ⅲ、Ⅳ期（P=0.000 1）。Western blotting显示在10组结直肠癌及配对癌旁组织中，CFB在结直肠癌组织中表达更高（P=0.000 2）。细胞划痕、Transwell小室结果显示，CFB促进RKO、SW620细胞的迁移、侵袭能力（P<0.01）。

结论

CFB在结直肠癌组织中高表达，并与不良预后相关，而RKO、SW620细胞的迁移、侵袭能力可被低表达的CFB抑制，提示其可作为结直肠癌患者预后的潜在标志物。

关键词: 结直肠肿瘤, 补体B因子, 计算生物, 迁移, 侵袭

Objective

To explore the expression and clinical significance of complement B factor (CFB) in colorectal cancer (CRC) based on bioinformatics and cell experiments.

Methods

HPA and TIMER 2.0 databases were used to analyze the expression of CFB in CRC tissue; GEPIA database analysis of the association between CFB and MKI67 expression in CRC. Kaplan-Meier Plotter database was used to analyze the relationship between the expression level of CFB mRNA and the survival of CRC patients; Gene enrichment pathway to predict the potential biological function of CFB in CRC; IHC and Western blotting validation of CFB expression in CRC tissue. The effects of CFB on migration and invasion of CRC cells were investigated by siRNA transfection, cell scratch and Transwell assay.

Results

The bioinformatics database showed that CFB was highly expressed in CRC tissues (P<0.001). The GEPIA database showed that CFB expression was higher in CRC tissues (P<0.05) and positively correlated with MKI67 expression (P=0.004 1). Kaplan-Meier Plotter survival analysis showed that low expression of CFB predicted poorer overall survival (P=0.015) and progression survival (P=0.048) in CRC. The enrichment results of CFB gene indicated that it might be involved in multiple progression pathways of CRC. IHC showed that the positive staining intensity of CFB in CRC tissue was higher than that in normal tissue, and TNM stageⅠand Ⅱ were weaker than Ⅲ and Ⅳ (P=0.000 1). Western blotting showed that in 10 cases of CRC and paired adjacent tissues, CFB was more expressed in cancer tissues (P=0.000 2). Cell scratch and Transwell cell results showed that CFB promoted the migration and invasion ability of RKO and SW620 cells (P<0.01).

Conclusion

CFB is highly expressed in CRC tissue and is associated with prognosis, while the migration and invasion ability of RKO and SW620 cells can be inhibited by low expression of CFB, indicating that it can serve as a potential prognostic marker for CRC.

Key words: Colorectal neoplasms, Complement B factor, Computational biology, Migration, Invasion

图1 研究补体B因子（CFB）在结直肠癌中表达及意义的实验设计流程图

表1 细胞siRNA转染序列

siRNA	正义链序列	反义链序列
阴性对照siRNA	5’-UUCUCCGAACGUGUCACGUTT-3’	5’-ACGUGACACGUUCGGAGAATT-3’
siRNA1	5’-CACGAAGCAGCUCAAUGAA-3’	5’-UUCAUUGAGCUGCUUCGUG-3’
siRNA2	5’- GGAGAUAGAAGUAGUCCUA-3’	5’-UAGGACUACUUCUAUCUCC-3’
siRNA3	5’- GUGCUAGAUGGAUCAGACA-3’	5’-UGUCUGAUCCAUCUAGCAC-3’

表1 细胞siRNA转染序列

siRNA	正义链序列	反义链序列
阴性对照siRNA	5’-UUCUCCGAACGUGUCACGUTT-3’	5’-ACGUGACACGUUCGGAGAATT-3’
siRNA1	5’-CACGAAGCAGCUCAAUGAA-3’	5’-UUCAUUGAGCUGCUUCGUG-3’
siRNA2	5’- GGAGAUAGAAGUAGUCCUA-3’	5’-UAGGACUACUUCUAUCUCC-3’
siRNA3	5’- GUGCUAGAUGGAUCAGACA-3’	5’-UGUCUGAUCCAUCUAGCAC-3’

图2 CFB在人体正常细胞、组织中的表达　A为CFB在细胞中的分布；B、C、D分别为CFB在结肠细胞、小肠细胞、不同人体组织中的表达水平

图3 CFB在不同肿瘤中的表达水平　A为CFB在泛癌水平上的表达，^* P<0.05，^** P<0.01，^*** P<0.001；B为结直肠癌组织与非癌组织中CFB表达的免疫组织化学结果（×100）

图4 CFB表达水平与结直肠癌患者预后的关系　A为CFB在结直肠癌组织中的表达，^* P<0.05；B为CFB与MKI67表达的相关性；C、D分别为结直肠癌患者的总生存率、无进展生存率

图5 结直肠癌中CFB基因的通路富集　A为生物过程的GO分析；B为细胞组成的GO分析；C为分子功能的GO分析；D为KEGG通路富集分析结果

表2 CFB表达与结直肠癌临床病理特点的关系(例)

项目	阳性	阴性	χ²值	P值
例数	35	28
性别			0.003	0.955
男	19	15
女	16	13
年龄(岁)			7.750	0.005
≥60	29	14
<60	6	14
病理分期			4.062	0.044
Ⅰ+Ⅱ	10	15
Ⅲ+Ⅳ	25	13
T分期			1.260	0.262
T1+T2	8	10
T3+T4	27	18

表2 CFB表达与结直肠癌临床病理特点的关系(例)

项目	阳性	阴性	χ²值	P值
例数	35	28
性别			0.003	0.955
男	19	15
女	16	13
年龄(岁)			7.750	0.005
≥60	29	14
<60	6	14
病理分期			4.062	0.044
Ⅰ+Ⅱ	10	15
Ⅲ+Ⅳ	25	13
T分期			1.260	0.262
T1+T2	8	10
T3+T4	27	18

图6 CFB在结直肠癌组织和癌旁正常组织中的表达情况　A、B分别为免疫组织化学、蛋白质水平检测结果；C为免疫组织化学联合染色评分比较；D为CFB蛋白的Western blotting检测量比较，^***P<0.001

图7 CFB对结直肠癌细胞迁移、侵袭的影响　A、B分别为RKO细胞、SW620细胞的Western blotting结果，^* P<0.05，^** P<0.01

图8 CFB对结直肠癌细胞迁移、侵袭的影响　A、D为RKO细胞Transwell小室实验结果（×40）；B、E为SW620细胞Transwell小室实验结果（×40）；C、F为结直肠癌细胞侵袭实验结果（×200）比较；^** P<0.01，^*** P<0.001

[1]	Sane S, Srinivasan R, Potts RA, et al. UBXN2A suppresses the Rictor-mTORC2 signaling pathway, an established tumorigenic pathway in human colorectal cancer[J]. Oncogene, 2023, 42(21): 1763-1776.
[2]	徐凤敏, 陈昱伶, 任玲, 等. S1P/S1PR1通路调节补体B因子的表达对食管鳞状细胞癌细胞增殖和迁移的影响[J]. 中国免疫学杂志, 2021, 37(19): 2364-2369.
[3]	Lu Q, Hou Q, Cao K, et al. Complement factor B in high glucose-induced podocyte injury and diabetic kidney disease[J]. JCI Insight, 2021, 6(19): e147716.
[4]	Wu P, Shi J, Sun W, et al. The prognostic value of plasma complement factor B (CFB) in thyroid carcinoma[J]. Bioengineered, 2021, 12(2): 12854-12866.
[5]	Shimazaki R, Takano S, Satoh M, et al. Complement factor B regulates cellular senescence and is associated with poor prognosis in pancreatic cancer[J]. Cell Oncol (Dordr), 2021, 44(4): 937-950.
[6]	Lee MJ, Na K, Shin H, et al. Early diagnostic ability of human complement factor B in pancreatic cancer is partly linked to its potential tumor-promoting role[J]. J Proteome Res, 2021, 20(12): 5315-5328.
[7]	Deng YZ, Yao F, Li JJ, et al. RACK1 suppresses gastric tumorigenesis by stabilizing the β-catenin destruction complex[J]. Gastroenterology, 2012, 142(4): 812-823, e15.
[8]	郭楠楠, 王兴智. 补体B因子与特发性膜性肾病的相关性研究[J]. 哈尔滨医科大学学报, 2022, 56(1): 21-25.
[9]	Di Stefano I, Alì G, Poma AM, et al. New immunohistochemical markers for pleural mesothelioma subtyping[J]. Diagnostics (Basel), 2023, 13(18): 2945.
[10]	Petr V, Thurman JM. The role of complement in kidney disease[J]. Nat Rev Nephrol, 2023, 19(12): 771-787.
[11]	Zanchi C, Locatelli M, Corna D, et al. Liver factor B silencing to cure C3 glomerulopathy: evidence from a mouse model of complement dysregulation[J]. Mol Immunol, 2023, 161: 25-32.
[12]	Akhlaghpour M, Haritunians T, More SK, et al. Genetic coding variant in complement factor B (CFB) is associated with increased risk for perianal Crohn’s disease and leads to impaired CFB cleavage and phagocytosis[J]. Gut, 2023, 72(11): 2068-2080.
[13]	Hakroush S,Tampe B. Association between loss of immune checkpoint programmed cell death protein 1 and active ANCA-associated renal vasculitis[J]. Int J Mol Sci, 2023, 24(3): 2975.
[14]	Robson JL, Thorn R, Williams AC, et al. Gut bacteria promote proliferation in benign S/RG/C2 colorectal tumour cells, and promote proliferation, migration and invasion in malignant HCT116 cells[J]. Sci Rep, 2023, 13(1): 17291.
[15]	Rodriguez-Hernandez I, Maiques O, Kohlhammer L, et al. WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion[J]. Nat Commun, 2020, 11(1): 5315.

[1]	罗青杉, 梅海涛, 郝家领, 蔡锦锋, 周润楷, 温玉刚. 连接蛋白43通过调控细胞周期抑制结直肠癌的增殖机制研究[J/OL]. 中华普通外科学文献(电子版), 2024, 18(05): 344-349.
[2]	马中正, 杨云川, 马翔, 周迟, 丁丁, 霍俊一, 徐楠, 崔培元, 周磊. 胰腺癌双硫死亡相关的lncRNA预后模型的构建及免疫反应研究[J/OL]. 中华普通外科学文献(电子版), 2024, 18(05): 368-376.
[3]	徐逸男. 不同术式治疗梗阻性左半结直肠癌的疗效观察[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 72-75.
[4]	谢丽春, 欧庆芬, 张秋萍, 叶升. 简化和标准肝脏MRI方案在结直肠癌肝转移患者随访中的临床应用[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(04): 434-437.
[5]	赵旭鹏, 王集琛, 田硕, 李宏召, 李修彬, 张旭. EP300 通过上调FKBP10 促进膀胱肿瘤细胞迁移和侵袭[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 264-274.
[6]	韩加刚, 王振军. 梗阻性左半结肠癌的治疗策略[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(06): 450-458.
[7]	梁轩豪, 李小荣, 李亮, 林昌伟. 肠梗阻支架置入术联合新辅助化疗治疗结直肠癌急性肠梗阻的疗效及其预后的Meta 分析[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(06): 472-482.
[8]	严虹霞, 王晓娟, 张毅勋. 2 型糖尿病对结直肠癌患者肿瘤标记物、临床病理及预后的影响[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(06): 483-487.
[9]	赵磊, 刘文志, 林峰, 于剑, 孙铭骏, 崔佑刚, 张旭, 衣宇鹏, 于宝胜, 冯宁. 深部热疗在改善结直肠癌术后辅助化疗副反应及生活质量中的作用研究[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(06): 488-493.
[10]	黄海洋, 邝永龙, 陈嘉胜. 基层医院结直肠肿瘤经自然腔道取标本手术30 例分析[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(06): 510-518.
[11]	任佳琪, 刁德昌, 何自衍, 张雪阳, 唐新, 李文娟, 李洪明, 卢新泉, 易小江. 网膜融合线导向的脾曲游离技术在左半结肠癌根治术中的应用[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(05): 362-367.
[12]	张迪, 王春霞, 张学东, 李发馨, 庞淅文, 陈一锋, 张维胜, 王涛. 梗阻性左半结直肠癌自膨式金属支架置入后行腹腔镜手术与开腹手术的短期临床疗效比较[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(05): 375-380.
[13]	张蔚林, 王哲学, 白峻阁, 黄忠诚, 肖志刚. 利用TCGA数据库构建基于miRNA的结直肠癌列线图预后模型[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(05): 381-388.
[14]	张伟伟, 陈启, 翁和语, 黄亮. 随机森林模型预测T1 期结直肠癌淋巴结转移的初步研究[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(05): 389-393.
[15]	王国强, 张纲, 唐建坡, 张玉国, 杨永江. LINC00839 调节miR-17-5p/WEE1 轴对结直肠癌细胞增殖、凋亡和迁移的影响[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 491-499.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Expression and clinical significance of complement B factor in colorectal cancer

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Expression and clinical significance of complement B factor in colorectal cancer