RCN3在结肠癌中的表达及其临床意义

The expression of RCN3 in colon cancer and its clinical significance

目的利用生物信息数据库结合生物实验分析RCN3(Reticulocalbin 3)在结肠癌中的表达及其临床意义。方法选取结肠癌HT29和SW620细胞以及结肠正常黏膜细胞FHC进行培养,采用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)实验验证RCN3在细胞中的表达情况。利用Ualcan数据库获取RCN3在正常结肠组织和结肠癌组织中的表达数据。从LinkedOmics数据库获取RCN3的共表达基因信息,通过基因本体论分析(GO)富集分析、京都基因与基因组百科全书(KEGG)通路分析确定这些RCN3共表达基因参与的生物学过程及相关功能。利用STRING数据库构建RCN3相关编码基因的蛋白互作网络。最后,根据GEPIA、Ualcan和LinkedOmics生物数据库共同对比分析RCN3的表达与结肠癌患者临床预后的关系。结果qRT-PCR和Western-blot实验结果显示HT29和SW620结肠癌细胞中RCN3的mRNA和蛋白表达水平均高于FHC正常细胞(均P<0.05)。通过分析生物数据库结果显示,结肠癌组织中RCN3的表达水平高于正常结肠组织(P<0.05)。GO富集分析显示:RCN3相关基因主要参与细胞外基质和细胞外区域结构的组成,细胞外基质与多种受体的结合过程以及通过细胞外区域结构参与如细胞黏附、免疫应答和血管生成等多种与肿瘤发展相关的生物过程。KEGG通路分析显示:RCN3相关基因主要参与了ECM受体相互作用、细胞因子受体的相互作用、趋化因子信号通路、PI3K-Akt信号通路、吞噬体信号、IgA相关肠道免疫网络信号等与肿瘤侵袭、迁移和炎症免疫反应相关的信号通路的传导。蛋白网络互作分析显示:与RCN3蛋白直接相互作用的编码蛋白基因主要包括了如PRDX6、NOSIP、PCSK6、IMMP1L、PRRG2、FBXO47、FCGRT、FKBP9、PCDHGA12、PNMAL1等,他们主要参与了结直肠癌、肝癌、胃癌、乳腺癌、肺癌、卵巢癌等多种肿瘤的发生发展进程。生存曲线分析显示,高表达RCN3的结肠癌患者的总生存率显著低于低表达的患者(P<0.05)。结论RCN3在结肠癌组织和细胞系中呈现高表达,且其与结肠癌的发生、发展及预后密切相关,可以作为结肠癌早期筛选、预后预测的标志物之一。

Objective To analyze the expression and clinical significance of reticulocalbin 3(RCN3)in colon cancer by bioinformatics database and biological experiments.Methods Colon cancer HT29 and SW620 cells and colon normal mucosal cells FHC were cultured.The expression of RCN3 in cells was verified by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot.The expression data of RCN3 in normal colon tissue and colon cancer tissue were obtained by Ualcan database.The co-expressed gene information of RCN3 from LinkedOmics database was obtained,and the biological processes and related functions of these RCN3 co-expressed genes through were analyzed by gene ontology analysis(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.The protein-protein interaction network of RCN3 related coding genes was constructed by using STRING database.Finally,the relationship between the expression of RCN3 and the clinical prognosis of patients with colon cancer was compared and analyzed according to GEPIA,Ualcan and Linked Omics biological database.Results Western blot and qRT-PCR results showed that the mRNA and protein expression of RCN3 in HT29 and SW620 colon cancer cells was significantly higher than those in FHCcells(all P<0.05).The analysis of biological database showed that the expression level of RCN3 in colon cancer tissue was higher than that in normal colon tissue(P<0.05).GO enrichment analysis showed that RCN3 co-expression genes were mainly involved in the composition of extracellular matrix and extracellular domain structure,the binding process of extracellular matrix and multiple receptors,and the biological processes related to tumor development such as cell adhesion,immune response,and angiogenesis through extracellular domain structure.KEGG pathway analysis showed that RCN3 co-expression genes were mainly involved in ECM receptor interaction,cytokine receptor interaction,chemokine signaling pathway,phosphatidylinositol-3-kinase protein kinase B(PI3K-Akt)signaling pathway,phagosome signal,IgA related intestinal immune network signal,these signaling pathways always related to tumor invasion,migration and inflammatory immune response.The protein-protein interaction network analysis showed that the coding protein genes that directly interacted with RCN3 protein that included PRDX6,NOSIP,PCSK6,IMMP1L,PRRG2,FBXO47,FCGRT,FKBP9,PCDHGA12,and PNMAL1,which were mainly involved in the occurrence and development of colorectal cancer,liver cancer,gastric cancer,breast cancer,lung cancer,and ovarian cancer.Survival curve analysis showed that the overall survival rate of colon cancer patients with high expression of RCN3 was significantly lower than that of patients with low expression of RCN3(P<0.05).Conclusions RCN3 is highly expressed in colon cancer tissues and cells,which is closely related to the occurrence,development and prognosis of colon cancer.It can be used as one of the markers for early screening and prognosis prediction of colon cancer.