基于在线数据库挖掘CLK3在结直肠癌中的表达差异及临床意义

Expression differences and clinical significance of CLK3 in colorectal cancer based on online database mining

目的:双特异性酪氨酸磷酸化调节激酶,也称CDC样激酶(cell division cycle like kinases,CDC-like kinases,CLK),是一种双特异性蛋白激酶,其参与神经系统疾病、代谢异常、肿瘤等多个病理及生理过程,其中CLK3参与肝癌、乳腺癌等多种肿瘤的发生和发展,但其在结直肠癌中的表达情况及临床意义尚无相关报道。本研究通过挖掘肿瘤在线数据库,探讨CLK3在结直肠癌中的表达及临床意义。方法:利用基因表达谱交互分析2(Gene Expression Profiling Interactive Analysis 2,GEPIA2)数据库及GEO2R工具对CLK3在肿瘤间及结直肠癌组织和正常组织间作差异分析,获取差异表达结果后利用UALCN(The University of Alabama at Birmingham Cancer Data Analysis Portal)数据库对CLK3表达差异组作Kaplan-Meier生存分析。利用LinkedOmics数据库对CLK3进行共表达分析,并筛选出与之呈正相关及负相关的各50个基因,然后对获取的100个基因进行基因本体(gene ontology,GO)和京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,同时利用TIMER2.0数据库对CLK3进行免疫相关分析。在组织表达层面,利用人类蛋白质图谱(Human Protein Atlas,HPA)数据库查询CLK3在结直肠癌与正常肠道组织的免疫组织化学标本,分析其表达差异。结果:在UALCN数据库中,与正常组织比较,CLK3在结肠癌组织中低表达(P<0.01),而在直肠癌组织中表达差异无统计学意义(P>0.05)。CLK3的表达水平与结直肠癌患者的预后显著相关(P<0.05),表现为其高表达与不良预后相关。GO与KEGG通路富集结果显示:CLK3共表达基因富集于抗原肽呈递、物质代谢合成过程、转录调节过程的酶反应、γ干扰素介导的信号通路、Wnt信号通路及肠道免疫炎症过程等。相关性分析显示:结肠癌中CLK3表达与BRAF、HERS、NTRK1、PIK3CA基因突变相关,直肠癌中CLK3表达与BRAF和PIK3CA基因突变相关。进一步免疫浸润分析结果显示:在结肠癌中,CLK3的表达水平与CD8+T细胞、CD4+T细胞、中性粒细胞和树突状细胞的浸润水平呈正相关(均P<0.05);在直肠癌中,CLK3的表达水平与CD4+T细胞、巨噬细胞、中性粒细胞和树突状细胞的浸润水平呈正相关(均P<0.05)。在免疫组织化学染色切片中,结肠癌和直肠癌CLK3蛋白表达水平均低于正常结肠组织(均P<0.05)。结论:CLK3在结直肠癌中存在差异表达,是结直肠癌生存预后的指标,有望成为结直肠癌的预后评估和临床治疗的有效靶点之一。

Objective:Dual-specificity tyrosine phosphorylation-regulated kinases(CDC-like kinases,CLKs)are a type of dual-specificity protein kinase involved in various pathological and physiological processes including neurological diseases,metabolic abnormalities,and tumors.Among them,CDC-like kinases 3(CLK3)is involved in the development of various cancers,such as hepatocellular carcinoma and breast cancer.However,its expression and clinical significance in colorectal cancer have not been reported.This study aims to explore the expression and clinical significance of CLK3 in colorectal cancer through mining online tumor databases.Methods:The differential expression of CLK3 between tumor tissues and normal tissues in colorectal cancer was analyzed by the Gene Expression Profiling Interactive Analysis 2(GEPIA2)database and the GEO2R tool.After obtaining the differential expression results,the University of Alabama at Birmingham Cancer Data Analysis Portal(UALCN)database was used for Kaplan-Meier survival analysis on the groups with differential expression of CLK3.The LinkedOmics database was used for co-expression analysis of CLK3,and the top 50 positively and negatively correlated genes were selected.These 100 genes were then subjected to gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.CLK3 was also analyzed for immune-related characteristics using the TIMER2.0 database.At the tissue expression level,the Human Protein Atlas(HPA)database was used to investigate the immunohistochemical specimens of CLK3 in colorectal cancer and normal intestinal tissues,analyzing its expression differences.Results:In the UALCN database,CLK3 was underexpressed in colon cancer tissues compared to normal tissues(P<0.01),while there was no significant difference in expression in rectal cancer tissues(P>0.05).The expression level of CLK3 was significantly associated with the prognosis of colorectal cancer patients(P<0.05),indicating that higher expression of CLK3 was associated with poor prognosis.GO and KEGG pathway enrichment results showed that genes co-expressed with CLK3 were involved in antigen peptide presentation,substance metabolism synthesis processes,enzyme reactions in transcription regulation processes,gamma interferon-mediated signaling pathways,Wnt signaling pathways,and intestinal immune inflammation processes.Correlation analysis showed that in colon cancer,the expression of CLK3 was associated with mutations in the BRAF,HERS,NTRK1,and PIK3CA genes,in rectal cancer,its expression was associated with BRAF and PIK3CA genes mutations.Further analysis of immune infiltration showed that in colon cancer,the expression level of CLK3 was positively correlated with the infiltration levels of CD8+T cells,CD4+T cells,neutrophils,and dendritic cells(all P<0.05),in rectal cancer,it was positively correlated with the infiltration levels of CD4+T cells,macrophages,neutrophils,and dendritic cells(all P<0.05).In immunohistochemical staining slides,the protein expression levels of CLK3 in both colon and rectal cancer were lower than those in normal colon tissues(all P<0.05).Conclusion:CLK3 exhibits differential expression in colorectal cancer and is an indicator of survival prognosis.It has the potential to become one of the effective targets for prognosis assessment and clinical treatment of colorectal cancer.