高通量技术检测抑癌基因Pitx2静默后PanIN细胞中下游基因改变

Identification of genes associated with malignant transformation in Pitx2-silenced PanIN cells through expression profiling analysis

目的:运用小鼠基因转录本的全基因表达谱芯片,研究Pitx2基因静默前后胰腺上皮内瘤变(pancreatic intraepithelial neoplasia,PanIN)细胞下游基因表达谱的变化。方法:应用小鼠全基因芯片检测Pitx2基因静默前后PanIN细胞基因表达谱的变化,并运用实时荧光定量多聚酶链反应(polymerase chain reaction,PCR)对部分芯片结果进行验证。结果:根据基因芯片筛选出差异倍数3倍以上的差异表达基因共97条。其中上调58条,涉及细胞增殖的基因包括Rac1、Ccr1、Fut9、PPP3CA、Sox5、Bmp7、Chtf18、Btg4、Flt4、Dscaml1和Pftk1;涉及细胞转移的基因包括Sesn1、Rpl7a、PPP3CA、Sox5、Bmp7、PIK3C2G、Cxcl12、Btg4和Kcnj14。下调39条,涉及细胞增殖的基因包括PPP2R2A、Nav1、PPP2R3C、Katnb1、Gpbp1、Nat3、Tgm5和PRKRA;涉及细胞转移的基因包括PPP2R2A、Lce1c、Hcn1、PPP2R3C、Tbxas1、Chrna1、Frmd5和Cdh7。其余基因涉及细胞周期、细胞死亡调控、细胞黏附、细胞转录活性、细胞转移酶活性。通过实时定量PCR对部分基因进行验证,显示基因芯片的结果与实时定量PCR验证结果基本一致。结论:通过基因芯片技术筛选出Pitx2基因静默前后PanIN细胞下游差异表达基因,为后续体内、外实验奠定了良好的基础。

Objective To explore the gene differentially expressed genes between pancreatic intraepithelial neoplasia(PanIN)cells and PanIN cells with Pitx2 silencing with gene microarray.Methods A gene microarray was performed to detect the differentially expressed genes in PanIN cells and PanIN cells with Pitx2 silencing.The differentially expressed genes were identified and subjected to real-time polymerase chain reaction(PCR)analysis.Results We identified 58 upregulated genes and 39 down-regulated genes that putatively resulted from Pitx2 knock-down in the PanIN cells.The upregulated genes associated with cell proliferation are Rac1,Ccr1,Fut9,PPP3CA,Sox5,Bmp7,Chtf18,Btg4,Flt4,Dscaml1 and Pftk1.The up-regulated genes associated with cell migration are Sesn1,Rpl7a,PPP3CA,Sox5,Bmp7,PIK3C2G,Cxcl12,Btg4 and Kcnj14.The down-regulated genes associated with cell proliferation are PPP2R2A,Nav1,PPP2R3C,Katnb1,Gpbp1,Nat3,Tgm5 and PRKRA.The down-regulated genes associated with cell migration are PPP2R2A,Lce1c,Hcn1,PPP2R3C,Tbxas1,Chrna1,Frmd5 and Cdh7.The other genes are involving in regulation of cell killing,cell cycle,cell adhesion,cell transferase activity and cell transcription.To validate the microarray results,several genes for real-time PCR confirmation were selected.For most genes,the magnitude of expression changes detected by PCR was comparable with the fold changes detected by the microarray platform.Conclusions The differentially expressed genes between PanIN cells and PanIN cells with Pitx2 silencing have been identified through gene expression profiling,faciliating further research.