【作者】 熊飞；

【导师】 陈勇军；

【作者基本信息】 华中科技大学 ， 外科学-胆胰外科， 2023， 博士

【摘要】 目的:探究HP1α蛋白对肝内胆管癌细胞增殖的调控作用及相关分子机制,并将该机制转化为治疗方案。方法:培养肝内胆管癌细胞系HUCCT1和HCCC-9810作为研究对象。利用免疫组化染色实验检测肿瘤及癌旁组织样品中HP1α蛋白的表达量,并利用平板克隆实验、CCK-8实验和流式细胞术评估HP1α表达量改变对肝内胆管癌细胞增殖的影响;利用实时定量PCR、Western Blot实验评估HP1α对I型干扰素通路的调控作用,并筛选出对应的调控靶点;利用蛋白质免疫共沉淀实验、染色质免疫共沉淀实验、转录组测序、电喷雾蛋白质谱技术和CUT&Tag技术来研究组蛋白乙酰化标志对STAT1基因表达的调控作用、HP1α和组蛋白去乙酰化酶之间的互作及该蛋白二聚体与STAT1基因启动子序列的相互作用;利用平板克隆实验、CCK-8实验和流式细胞术评估组蛋白去乙酰化酶抑制剂TSA和干扰素制剂对肝内胆管癌细胞增殖的抑制作用,并在小鼠肝内胆管癌模型和裸鼠皮下瘤模型中进行验证。结果:HP1α在肝内胆管癌组织中高表达,敲减HP1α显著抑制了肝内胆管癌细胞的增殖。敲减HP1α后,STAT1和干扰素刺激基因的m RNA水平和蛋白水平显著上调,I型干扰素通路活化水平升高。STAT1基因的表达量受到组蛋白乙酰化标志的调控,特别是H3K27ac标志。HP1α与HDAC1形成蛋白二聚体,富集于STAT1基因的启动子区域,诱导局部H3K27ac标志去乙酰化,并上调局部H3K9me3标志的含量。TSA药物处理一方面下调了HP1α的表达量,另一方面减少了HP1α-HDAC1复合体在STAT1启动子特定区域的富集。使用TSA和干扰素制剂分别处理肝内胆管癌细胞后,细胞增殖受到显著抑制。相比于干扰素制剂单药治疗,联合应用干扰素制剂和TSA对肝内胆管癌细胞的增殖抑制作用更加显著。HP1α参与介导肝内胆管癌细胞对两种药物的抵抗作用。结论:HP1α-HDAC1复合体通过直接结合至STAT1基因的启动子区域,影响局部环境H3K27ac和H3K9me3组蛋白标志的含量,转录下调STAT1的表达量,抑制I型干扰素信号通路的活化,促进肝内胆管癌细胞的增殖。干扰素制剂和组蛋白去乙酰化酶抑制剂TSA联合应用方案通过靶向HP1α-HDAC1复合体,抑制肝内胆管癌细胞的增殖,可以为肝内胆管癌的诊疗实践和药物开发提供一定的参考。更多还原

【Abstract】 Objective: To investigate the regulatory effect of HP1α protein on the proliferation of intrahepatic cholangiocarcinoma cells and the related molecular mechanism,and to transform the mechanism into a feasible therapeutic regimen.Methods: Intrahepatic cholangiocarcinoma cell line HUCCT1 and HCCC-9810 were cultured.Immunohistochemical assay was used to measure the expression level of HP1α in tumor and adjacent tissue samples.The effect of HP1α expression change on the proliferation of intrahepatic cholangiocarcinoma cells was evaluated by colony formation assay,CCK-8 assay and flow cytometry.Real-time quantitative PCR and Western Blot were used to evaluate the regulatory effect of HP1α on Type I Interferon signaling pathway.The corresponding regulatory target was then screened out.Protein immunoprecipitation assay,chromatin immunoprecipitation assay,RNA sequencing,electrospray ionization mass spectrometry and CUT&Tag assay were used to investigate the function of histone acetylation markers,the interaction between HP1α and histone deacetylase and the interaction between the protein dimer and STAT1 promoter region.The inhibitory effects of histone deacetylase inhibitor TSA and interferon preparation on the proliferation of intrahepatic cholangiocarcinoma cells were evaluated by colony formation assay,CCK-8 assay and flow cytometry.The conclusion was further verified in murine intrahepatic cholangiocarcinoma model and nude mice model.Results: HP1α was found to be highly expressed in intrahepatic cholangiocarcinoma tissue.HP1α knockdown can significantly inhibit the proliferation of intrahepatic cholangiocarcinoma cells.After HP1α was knocked down,the m RNA level and protein level of STAT1 and interferon-stimulated genes were significantly up-regulated and the Type I Interferon signaling pathway was activated.STAT1 can be regulated by histone acetylation markers,especially H3K27 ac.HP1α can form protein dimer with HDAC1 and be located in the promoter region of STAT1 gene.HP1α-HDAC1 complex can down-regulate H3K27 ac markers,and up-regulate H3K9me3 markers in local environment.TSA treatment down-regulated the expression of HP1α and reduced the distribution of HP1α-HDAC1 complex in the specific region of STAT1 promoter.Interferon preparation treatment and TSA treatment both exhibited significant inhibition on the proliferation of intrahepatic cholangiocarcinoma cells.Compared with interferon preparation monotherapy,the combination of interferon preparation and TSA had stronger inhibiting effects on the proliferation of intrahepatic cholangiocarcinoma cells.Conclusions: HP1α-HDAC1 complex affects the distribution of histone markers,e.g.,H3K27 ac and H3K9me3 by directly binding to the promoter region of STAT1 gene,transcriptionally down-regulating the expression level of STAT1 and inhibiting the Type I Interferon pathway.Combined application of interferon preparation and TSA can inhibit the proliferation of intrahepatic cholangiocarcinoma cells by targeting HP1α-HDAC1 complex,which can provide a certain reference for the clinical practice and drug development of intrahepatic cholangiocarcinoma treatment.更多还原