基于芯片技术探讨结直肠癌中沉默DNA结合蛋白A的基因表达谱分析

Gene expression profile analysis on silencing DNA binding protein A in colorectal cancer based on chip technology

目的基于基因芯片技术探讨短发夹RNA沉默人结直肠癌细胞DNA结合蛋白A(dbpA)表达后利用基因本体(GO)和京都基因与基因组百科全书(KEGG)分析发现与dbpA相关的结直肠癌发生、发展的信号通路。方法选取结直肠癌SW620细胞中表达差异的基因进行深度检测和分析,采用基因芯片技术挖掘差异基因,并运用定量反转录聚合酶链反应(RT-PCR)对芯片结果进行验证,综合分析工具进行GO功能富集分析及KEGG信号通路分析。结果dbpA敲低后基因芯片检测差异表达基因有578条,其中181个基因上调,397个基因下调,显著上调基因有CFL2、C12orf39、EREG、SESN2、CXCL3、CXCL1、VLDVR、DDIT4、INHBE等60个基因,显著下调基因在PRDM10、PCDH19、SRSF8、FGFBP1、TFF2、MTDH、TCF7、EHHADH、WDR77、CEACAM6等60个基因。dbpA敲除后SW620细胞中与肿瘤形成有关的通路主要与“生物过程”有关,包括丝裂原活化蛋白激酶信号通路、癌症途径、NOD样受体信号通路、趋化因子信号通路、粘着斑、基底细胞癌、Wnt信号通路等。基因网络图分析显示有10个基因表达水平下降(FASLG、PDGFA、FGF18、MAP3K7、FGF3、CSDA、NF1、RAP1A、HSPA1A、LAMTOR3等),4个基因表达水平上升(DUSP5、PRAS2、CDC42、DUSP2等),dbpA与MAPK信号通路相关性最大。选择KMAPK信号通路中的5个相关基因即RAPIA、TAK1(MAP3K7)、DUSP5、CDC42、ZAK进行Western blot验证表明,DUSP5表达水平明显上调(P<0.05),RAP1A、TAK1、ZAK表达水平下降,RAP1A、TAK1表达水平下降明显(P<0.05)。dbpA可能通过MAPK通路调节结直肠癌细胞的发生。结论dbpA参与调控的靶基因和信号通路可能为MAPK相关通路,还需要进一步在多细胞和动物实验中研究揭示dbpA下调通路之间的关系及CDC42在结直肠癌发展中的作用。

Objective To investigate to use the gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)assay to find dbpA-related signaling pathways for the occurrence and development of colorectal cancer after shRNA silencing colorectal cancer cells dbpA expression based on the chip technology.Methods The differentially expressed genes in colorectal cancer SW620 cells were selected for in-depth detection and analysis,and the differentially expressed genes were extracted by using gene chip technology,and the chip results were verified by q-PCR.The GO function enrichment analysis and KEGG signaling pathway analysis were performed by the comprehensive analysis tools.Results After dbpA knockdown,there were 578 differentially expressed genes detected by gene chip,in which 181 genes were up-regulated and 397 genes were down-regulated,the significantly up-regulated genes included 60 genes of CFL2,C12orf39,EREG,SESN2,CXCL3,CXCL1,VLDVR,DDIT4,INHBE,etc.The significantly down-regulated genes included 60 genes of PRDM10,PCDH19,SRSF8,FGFBP1,TFF2,MTDH,TCF7,EHHADH,WDR77,CEACAM6,etc.After dbpA knockout,the tumor formation related pathways in SW620 cells were mainly related to“biological processes”,including the MAPK signaling pathway,pathways in cancer,NOD like receptor signaling pathway,chemokine signaling pathway,focal adhesion,basal cell carcinoma and Wnt signaling pathway,etc.The gene network analysis showed that the expression levels of 10 genes(FASLG,PDGFA,FGF18,MAP3K7,FGF3,CSDA,NF1,RAP1A,HSPA1A,LAMTOR3)were decreased,and the expression levels of 4 genes(DUSP5,PRAS2,CDC42,DUSP2)were increased.The correlation between dbpA and MAPK signaling pathway was found to be the maximal.The five genes related to MAPK signaling pathway,i.e.RAPIA,TAK1(MAP3K7),DUSP5,CDC42 and ZAK,were selected for conducting the Westernblot verification,which indicated that the DUSP5 expression level was significantly up-regulated(P<0.05).The expression levels of RAP1A,TAK1 and ZAK were decreased,which of RAP1A and TAK1 were decreased significantly.dbpA could regulate the occurrence of colorectal cancer cells possibly through MAPK pathway.Conclusion The target genes and signali further studies in multi-cell and animal experiments are needed to reveal the relationship between dbpA down-regulation pathways and the role of CDC42 in the development of colorectal cancer.