CAJ | 학술논문

目的探讨肝细胞核因子1α(HNF1α)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HNF1α的表达,基因芯片技术检测处理后细胞的表达谱,筛选受HNF1α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HNF1α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HNF1α对TGFβ信号通路相关基因的调控作用.结果在HuH7细胞中,携带HNF1α基因的慢病毒Lenti-HNF1α可显著上调HNF1α表达,而含有静默HNF1α表达片段的慢病毒Lenti-shHNF1α可下调其表达.基因芯片结果显示,受HNF1α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,HNF1α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-κB、p53等肿瘤相关信号通路.荧光素酶报告基因实验表明,上调HNF1α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HNF1α能够抑制TGFβ信号通路相关基因c-myc和TGFβ1基因的表达,从而抑制该信号通路的活化.结论 HNF1α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HNF1α能够抑制TGF-β信号通路的活化.
목적 탐토간세포핵인자1α(HNF1α)대간암HuH7세포기인표체보적영향급기대상관신호통로적조공작용.방법 이용만병독개도상조혹하조HuH7세포중HNF1α적표체,기인심편기술검측처리후세포적표체보,사선수HNF1α조공적차이기인병이용DAVID연건화상관분석계통진행신호통로귀속분석;보고기인실험검측HNF1α대TGFβ신호통로적조공작용,동시이용실시형광정량PCR화Western인적법검측HNF1α대TGFβ신호통로상관기인적조공작용.결과 재HuH7세포중,휴대HNF1α기인적만병독Lenti-HNF1α가현저상조HNF1α표체,이함유정묵HNF1α표체편단적만병독Lenti-shHNF1α가하조기표체.기인심편결과현시,수HNF1α정향조공차이2배이상적기인유339개,부향조공차이2배이상적기인유325개.차이기인신호통로귀속분석표명,HNF1α능구조공약물대사、불포화지방산적합성、당효해/당이생등대사상관신호통로,동시환삼여조공TGFβ、NF-κB、p53등종류상관신호통로.형광소매보고기인실험표명,상조HNF1α능구억제TGFβ신호통로활화;동시실시형광정량PCR화Western인적법증실,상조HNF1α능구억제TGFβ신호통로상관기인c-myc화TGFβ1기인적표체,종이억제해신호통로적활화.결론 HNF1α대HuH7세포적기인표체보화종류발생、발전상관신호통로구유엄범영향,차HNF1α능구억제TGF-β신호통로적활화.
Objective To investigate the regulation effect of hepatocyte nuclear factor (HNF) 1α on the gene expression profile and the signal pathways in HuH7 cells.Methods The expression of HNF1α was increased or decreased in HuH7 cells by Lenti-virus carrying HNF1α or shHNF1α.The expression profile of the cells after treated was examined by microarray technology.The difference expressed gene regulated by HNF1α were screened and the pathway was analyzed with DAVID software and related analysis system.The regulation effect of HNF1α on transforming growth factor (TGF)β signal pathway was detected by reporter gene test and the regulation role of HNF1α on related genes of TGFβ signal pathway was determined by real-time polymerase chain reaction (PCR) and Western blotting assay.Results The expression of HNF1α in HuH7 cells was significantly up-regulated by Lenti-virus carrying HNF1α gene (Lenti-HNF1α) and which was down regulated by Lenti-virus with shHNF1α gene (LentishHNF1 α).Expression profile analysis revealed that 339 genes were positively up regulated two times by HNF1α and 325 genes were negatively down regulated two times.Signal pathway analysis revealed that HNF1α 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)-κB and p53 tumor-related signal pathways.Furthermore,Luciferase reportor gene experiment indicated that up-regulated HNF1α could inhibit the activation of TGFβ signal pathway.And the results of real-time PCR and Western blotting verified that up-regulated HNF1α could inhibit TGFβ signal pathway related gene c-myc and TGFβ1 and then inhibited the activation of TGFβ signal pathway.Conclusion HNF1α broadly affects the gene expression profile and the tumor genesis and development related signal pathways in HuH7 cells,furthermore,HNF1α can inhibit the activation of TGFβ signal pathway.