Y-box结合蛋白1通过Gli2调控肝祖细胞增殖  

Y-box binding protein-1 modulates hepatic progenitor cells proliferation via Gli2

作　　者：李飞[1] 李兵航 郭悦承 沈波 顾天翊 曲颖[1] 张启迪[1] 蔡晓波[1] 陆伦根[1] LI Fei;LI Bing-hang;GUO Yue-cheng;SHEN Bo;GU Tian-yi;QU Ying;ZHANG Qi-di;CAI Xiao-bo;LU Lun-gen(Department of Gastroenterology,Shanghai First People′s Hospital,Shanghai Jiao Tong University,Shanghai 20080,China)

机构地区：[1]上海交通大学附属第一人民医院消化科,200080

出　　处：《肝脏》2021年第5期557-562,共6页Chinese Hepatology

基　　金：国家自然科学基金(81800509)。

摘　　要：目的探索Y-box结合蛋白1(YB-1)对肝祖细胞(HPC)增殖的影响及机制。方法慢病毒载体系统下调YB-1的表达,细胞增殖实验和流式细胞术检测细胞增殖和细胞周期;RNA测序分析YB-1对HPC表达谱的影响;染色质免疫共沉淀结合高通量测序(ChIP-sequence)检测YB-1在HPC中的靶基因;实时PCR和蛋白质印迹检测相关基因表达;ChIP-PCR验证YB-1与Gli2启动子结合;荧光素酶报告基因实验分析YB-1调控Gli2的转录;Gli1/Gli2阻滞剂与HPC共培养并检测细胞增殖。结果YB-1 mRNA下调67%和75%后HPC增殖率分别下降58%和70%;RNA-sequence显示YB-1能调控Hedgehog信号通路、Wnt信号通路以及MAPK信号通路;ChIP-sequence显示YB-1在HPC中的靶基因参与细胞粘附、Wnt信号通路、Hedgehog信号通路,且YB-1能结合于Gli2启动子区;ChIP-PCR和荧光素酶报告基因实验表明YB-1能与Gli2结合并正调控其转录;实时PCR和蛋白质印迹证实YB-1可以调控Hedgehog信号通路上多种信号分子的表达;与对照组相比,Gli1/Gli2阻滞剂处理后处于有丝分裂期的HPC细胞数下降60%以上。结论YB-1能与Gli2启动子区结合并正向调控其转录,进而调控HPC增殖。Objective To explore the effect of Y-box binding protein-1(YB-1)on the proliferation of hepatic progenitor cells(HPCs)and the molecular mechanism.Methods The lentiviral vector system was adopted to down-regulate YB-1 expression.Cell counting kit 8(CCK-8)and flow cytometry were performed to assess the proliferation rate.RNA-sequencing was employed to evaluate the effect of YB-1 on gene expression of HPCs.Chromatin immunoprecipitation coupled with high-throughput deep sequencing(ChIP-Sequence)was performed to detect the potential target genes of YB-1 in HPCs.ChIP-PCR was conducted to identify the binding of YB-1 and Gli2.Luciferase reporter assay was conducted to analyze the regulatory effect of YB-1 on Gli2 transcription.Gli1/Gli2 inhibitor GANT61 was co-culture with HPCS to assess the effect of Gli2 on HPCs proliferation.Results When YB-1 mRNA expression down-regulated by 67%and 75%,respectively,HPCs proliferation decreased by 58%and 70%.RNA-sequence demonstrated that YB-1 involved in regulation of TGF-beta signaling pathway,Hedgehog signaling pathway,Wnt signaling pathway and MAPK signaling pathway.ChIP-sequence indicated the target gene of YB-1 participated in MAPK signaling pathway,Wnt signaling pathway,Hedgehog signaling pathway.Real-time PCR and Western blotting identified down-regulation of YB-1 depressed the expression of several molecules involving in the Hedgehog signaling pathway.ChIP-PCR and luciferase reporter assay showed that YB-1 could bind to the promoter of Gli2 and modulated the transcription.CCK-8 and EdU assay indicated that Gli-1/Gli2 inhibitor GANT61 could depress HPCs proliferation up to 60%.Conclusion YB-1 can bind to the promoter of Gli2 and modulates the transcription of Gli2 further to regulate the proliferation of hepatic progenitor cells.

关 键 词：YB-1 GLI2 肝祖细胞 增殖 

分 类 号：R73[医药卫生—肿瘤]