Y-box结合蛋白1通过Gli2调控肝祖细胞增殖

肝脏 ›› 2021, Vol. 26 ›› Issue (5): 557-562.

Y-box结合蛋白1通过Gli2调控肝祖细胞增殖

李飞, 李兵航, 郭悦承, 沈波, 顾天翊, 曲颖, 张启迪, 蔡晓波, 陆伦根

200080 上海交通大学附属第一人民医院消化科

收稿日期:2020-12-31 出版日期:2021-05-31 发布日期:2021-06-17
通讯作者: 陆伦根,Email:lungenlu1965@163.com
基金资助:
国家自然科学基金(81800509)

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

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

Received:2020-12-31 Online:2021-05-31 Published:2021-06-17
Contact: LU Lun-gen, Email: lungenlu1965@163.com

摘要/Abstract

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。

关键词: YB-1, Gli2, 肝祖细胞, 增殖

Abstract: 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.

Key words: Y-box binding protein-1 (YB-1), Gli2, Hepatic progenitor cells, Proliferation

李飞, 李兵航, 郭悦承, 沈波, 顾天翊, 曲颖, 张启迪, 蔡晓波, 陆伦根. Y-box结合蛋白1通过Gli2调控肝祖细胞增殖[J]. 肝脏, 2021, 26(5): 557-562.

LI Fei, LI Bing-hang, GUO Yue-cheng, SHEN Bo, GU Tian-yi, QU Ying, ZHANG Qi-di, CAI Xiao-bo, LU Lun-gen. Y-box binding protein-1 modulates hepatic progenitor cells proliferation via Gli2[J]. Chinese Hepatolgy, 2021, 26(5): 557-562.

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