摘要
目的探讨肝细胞核因子la（HNFla）对肝癌HuH7细胞基因表达谱的影响及其对相关信号通路的调控作用。方法利用慢病毒介导上调或下调HuH7细胞中HNFβ的表达，基因芯片技术检测处理后细胞的表达谱，筛选受HNFla调控的差异基因并利用DAVID软件和相关分析系统进行信号通路归属分析；报告基因实验检测HNFla对TGFβ信号通路的调控作用，同时利用实时荧光定量PCR和Western印迹法检测HNFla对TGFβ信号通路相关基因的调控作用。结果在HuH7细胞中，携带HNFla基因的慢病毒Lenti—HNFld可显著上调HNFla表达，而含有静默HNFla表达片段的慢病毒Lenti—shHNFld可下调其表达。基因芯片结果显示，受HNFla正向调控差异2倍以上的基因有339个，负向调控差异2倍以上的基因有325个。差异基因信号通路归属分析表明，HNFla能够调控药物代谢、不饱和脂肪酸的合成、糖酵解／糖异生等代谢相关信号通路，同时还参与调控TGFβ、NF—xB、p53等肿瘤相关信号通路。荧光素酶报告基因实验表明，上调HNFβ能够抑制TGFβ信号通路活化；同时实时荧光定量PCR和Western印迹法证实，上调HNFla能够抑制TGFβ信号通路相关基因c—myc和TGFgl基因的表达，从而抑制该信号通路的活化。结论HNFβ对HuH7细胞的基因表达谱和肿瘤发生、发展相关信号通路具有广泛影响，且HNFla能够抑制TGF-β信号通路的活化。
Objective To investigate the regulation effect of hepatocyte nuclear factor （HNF） la on the gene expression profile and the signal pathways in HuH7 cells. Methods The expression of HNFβ was increased or decreased in HuH7 cells by Lenti-virus carrying HNFla or shHNFla. The expression profile of the cells after treated was examined by microarray technology. The difference expressed gene regulated by HNFla were screened and the pathway was analyzed with DAVID software and related analysis system. The regulation effect of HNFβ on transforming growth factor （TGF）β signal pathway was detected by reporter gene test and the regulation role of HNFla on related genes of TGFβ signal pathway was determined by real-time polymerase chain reaction （PCR） and Western blotting assay. Results The expression of HNFla in HuH7 cells was significantly up-regulated by Lenti-virus carrying HNFla gene （Lenti-HNFla） and which was down regulated by Lenti-virus with shHNFla gene （Lenti- shHNFla）. Expression profile analysis revealed that 339 genes were positively up regulated two times by HNFla and 325 genes were negatively down regulated two times. Signal pathway analysis revealed that HNFla regulated drug metabolism, biosynthesis of unsaturated fatty acids and glycolysis/gluconeogenesis metabolism signal pathways. Moreover, it also involved in the regulation of TGFβ, nuclear factor （NF）-kB and p53 tumor-related signal pathways. Furthermore, Luciferase reportor gene experiment indicated that up-regulated HNFla could inhibit the activation of TGFβ signal pathway. And the results of real-time PCR and Western blotting verified that up-regulated HNFla could inhibit TGFβ signal pathway relatedgene c-myc and TGFβ and then inhibited the activation of TGFβ signal pathway. Conclusion HNFla broadly affects the gene expression profile and the tumor genesis and development related signal pathways in HuH7 cells, furthermore, HNFla can inhibit the activation of TGFβ signal pathway.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2015年第3期193-197,共5页
Chinese Journal of Digestion
基金
国家自然科学基金（81372675）
关键词
肝细胞核因子1a
基因表达谱
TGFβ信号通路
Hepatocyte nuclear factor 1-alphas Gene expression profiling TGF-β signal pathway
