【作者】 王海燕；

【导师】 陶永光；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2023， 博士

【摘要】 研究目的:细胞生存和死亡间的平衡是机体存活的重要条件,不同的细胞命运间存在着相互联系。铁死亡是一种由铁依赖的脂质过氧化作用驱动的程序性细胞死亡,研究表明铁死亡和自噬、凋亡间存在相互交流,但铁死亡和焦亡两种细胞死亡间的交互关系及其机制尚不清楚。因此,我们开展此课题拟探讨细胞铁死亡和焦亡间的交互关系及其关键调控分子,进一步探索该分子的上游调控基因对细胞铁死亡和焦亡的影响。本研究将为肝癌诊断和治疗提供新的分子靶点,期望为进一步表征肿瘤异质性提供新的理论依据,具有重要的研究意义。研究方法:(1)为探究铁死亡和焦亡间的相互交流,我们首先用聚丙烯塑料微粒、铁死亡诱导剂RSL3、铁死亡抑制剂(ferrostatin-1、liproxstatin-1)、焦亡诱导剂(LPS和Nigericin、CHX和TNFα)处理肝细胞或肝癌细胞,并检测细胞活力、LDH释放量。此外,铁死亡诱导剂RSL3或ML162处理敲降焦亡核心基因GSDMD或GSDME的肝癌细胞,检测细胞活力、lipid ROS含量。(2)为探究调控铁死亡和焦亡间相互交流的关键基因,我们首先采用单样本基因集富集分析(ss GSEA)从4个GEO数据集中筛选出同时与铁死亡和焦亡评分相关的基因。随后采用铁死亡诱导剂RSL3、铁死亡抑制剂Fer-1、焦亡诱导剂(LPS和Nigericin、CHX和TNFα)处理肝癌细胞,Western blot实验检测铁死亡和焦亡相关蛋白。随后采用光学显微镜观察细胞死亡形态,试剂盒检测LDH释放量,CCK8实验检测细胞活力探究HMGCR对细胞焦亡和铁死亡的影响。此外,采用免疫荧光实验和抽提线粒体蛋白实验验证HMGCR在线粒体和内质网的定位情况。(3)为探究HMGCR上游调控机制,我们首先在工具细胞293T中分别转染多种DUB质粒筛选出上调HMGCR表达的DUB,接着利用免疫荧光和Co-IP实验探究BRCC36与HMGCR两者间是否存在相互作用。随后采用CHX及MG132处理过表达或敲除BRCC36的肝癌细胞系,并进行去泛素化实验,探究BRCC36是否可去泛素化修饰HMGCR。(4)为探究BRCC36对肝癌细胞铁死亡的影响,我们采用CCK8实验、流式细胞术实验和透射电镜实验研究BRCC36对细胞铁死亡影响。采用CCK8实验检测细胞活力,电镜观察细胞死亡形态,试剂盒检测LDH释放量,Western blot实验检测焦亡相关蛋白探究BRCC36对细胞焦亡的影响。随后在敲除BRCC36的肝癌细胞系中过表达HMGCR,通过CCK8实验和LDH释放实验探究BRCC36是否通过调控HMGCR影响肝癌细胞铁死亡和焦亡。(5)为探究BRCC36在肝癌中发挥的作用,我们首先利用生信数据库、免疫组化实验分析BRCC36在肝癌中的表达水平,进一步采用CCK8实验、平板克隆实验、Transwell迁移侵袭实验、裸鼠成瘤实验研究BRCC36对肝癌细胞增殖、迁移能力的影响。此外,通过泛素化实验、CCK8实验、克隆形成等实验检测BRCC36抑制剂Thiolutin对肝癌恶性进展及肝癌细胞铁死亡和焦亡的影响。研究结果:(1)细胞铁死亡和焦亡间存在相互拮抗关系,铁死亡抑制剂Fer-1可以增加焦亡诱导剂诱导的细胞死亡和LDH释放。此外,敲降GSDMD或GSDME促进铁死亡诱导剂RSL3诱导的细胞死亡和脂质过氧化物的累积。(2)HMGCR是调节这两种死亡模式间相互交流的关键分子,其促进细胞焦亡和抑制铁死亡。当用铁死亡诱导剂处理细胞后,HMGCR主要定位于线粒体;当用焦亡诱导剂处理细胞后,HMGCR主要定位于内质网。(3)BRCC36作为HMGCR的去泛素化酶,其主要通过移除K63位多聚泛素链稳定HMGCR表达,而BRCC36的122和124位组氨酸突变为谷氨酰胺可增加HMGCR泛素化水平。(4)BRCC36抑制RSL3诱导的细胞死亡和脂质过氧化物的蓄积,而且增加细胞发生焦亡的数量和LDH的释放。此外,BRCC36通过调控HMGCR抑制细胞铁死亡和促进焦亡。(5)BRCC36在肝癌组织中高表达,与患者预后有关,并促进肝癌细胞增殖迁移。BRCC36抑制剂硫藤黄菌素显著抑制BRCC36与HMGCR的相互作用,抑制肝癌细胞增殖,加剧RSL3诱导的细胞死亡。研究结论:(1)HMGCR调控铁死亡和焦亡间的相互交流,且BRCC36是HMGCR的去泛素化酶。(2)BRCC36抑制细胞铁死亡和促进细胞焦亡,且通过调控HMGCR影响细胞铁死亡和焦亡。在肝癌细胞中,BRCC36促进细胞增殖迁移,靶向BRCC36抑制肝癌进展,揭示BRCC36可以作为肝癌治疗的潜在靶点。图43幅,表19,参考文献133篇更多还原

【Abstract】 Background:The balance between cell survival and cell death is critical for organism survival,and there are interactions between diverse cell death.Ferroptosis is an iron-dependent cell death triggered by the accumulation of lipid peroxide in cellular membranes.Ferroptosis,which is related to autophagy and apoptosis,may interact with pyroptosis in an unknown way.As a result,we carried out this research to explore the interplay between ferroptosis and pyroptosis,identify an important regulator in this interaction,and the upstream genes of this regulator.This study could provide a new reference for future characterization of tumor heterogeneity in targeted therapy,and provide novel molecular targets and therapeutic strategies for liver cancer treatment,which has substantial clinical implications.Methods:(1)To explore the interplay between ferroptosis and pyroptosis,the liver cells or liver cancer cells were treated with polystyrene microplastics,ferroptosis inducers RSL3,ferroptosis inhibitors(ferrostatin-1,liproxstatin-1),pyroptosis inducers(LPS and nigericin,CHX and TNFα),and the cell viability and LDH release were detected.Moreover,the knockdown GSDMD/GSDME of hepatocellular carcinoma cells were treated with ferroptosis inducers RSL3 or ML162,and the cell viability and lipid ROS level were measured.(2)To identify critical regulator in the interplay between ferroptosis and pyroptosis,we first utilized single sample gene set enrichment analysis(ss GSEA)from four GEO datasets to screen the genes associated with both ferroptosis and pyroptosis.Liver cancer cells were treated with RSL3,the inhibitor Fer-1 or pyroptosis inducer(LPS and Nigericin,CHX and TNFα),and Western blot assay was performed.To investigate the effect of HMGCR on ferroptosis and pyroptosis,we detected the morphologic number of cell death by using electron microscopy,LDH release by using a LDH kit and the cell viability by using CCK8 assay.Moreover,immunofluorescence(IF)and mitochondrial protein extraction assay to detect the HMGCR localization in mitochondria and endoplasmic reticulum.(3)To evaluate the upstream regulation mechanism of HMGCR,we first transfected several DUBs into 293 T cells to screen for DUBs that increased HMGCR expression.We next performed IF and Co-IP assays to determine whether there was an interaction between BRCC36 and HMGCR.Moreover,the liver cancer cell lines with overexpression or knockout of BRCC36 were treated with CHX or MG132,and deubiquitylation tests were carried out to determine if BRCC36 could function as a DUB of HMGCR.(4)To explore the impact of BRCC36 on ferroptosis in liver cancer cells,we used the CCK8 assay,flow cytometer assay,and transmission electron microscopy assay.To investigate the effect of BRCC36 on pyroptosis,cell death morphology was observed using an electron microscope,LDH release was measured using a kit,and pyroptosisrelated proteins were determined by using Western blot assay.Moreover,HMGCR was overexpressed in BRCC36-Knockout liver cancer cells,and CCK8 and LDH release assays were performed to determine whether BRCC36 affected ferroptosis and pyroptosis via regulation of HMGCR.(5)To study the function of BRCC36 in liver cancer,we first analyzed the expression level of BRCC36 using a bioinformatics database and immunohistochemical staining assay.The CCK8 assay,colony formation assay,transwell migration and invasion assay,and tumor formation assay were used to evaluate the impact of BRCC36 on the proliferation and migration of liver cancer cells.Finally,the ubiquitination assay,CCK8 assay,and the colony formation assay were used to determine the effect of thiolutin,a BRCC36 inhibitor,on the malignant progression of liver cancer.Results:(1)We demonstrated that the relationship between ferroptosis and pyroptosis was mutually antagonistic.Fer-1 increased pyroptosis inducertriggered cell death and LDH release.Furthermore,the GSDMD or GSDME knockdown promoted cell death and lipid peroxide accumulation triggered by RSL3 or ML162.(2)HMGCR was a key factor in the interaction between these two modes,and HMGCR promoted pyroptosis and inhibited ferroptosis.We further found that HMGCR mainly located in mitochondria when cells underwent ferroptosis,and HMGCR chiefly located in the endoplasmic reticulum after pyroptosis inducer treatment.(3)As the HMGCR de-ubiquitination enzyme,BRCC36 primarily removed the K63 polyubiquitin chain to stabilize HMGCR expression,whereas the BRCC36 mutation increased the ubiquitination level of HMGCR.(4)BRCC36 reduced RSL3-induced cell death and lipid ROS accumulation,but increased cell death and LDH release.BRCC36 significantly inhibited ferroptosis and promoted pyroptosis through deubiquitinating HMGCR.(5)BRCC36 was highly expressed in liver cancer,and the elevated BRCC36 expression had shorter overall survival compared with low BRCC36 expression.BRCC36 promoted the viability of proliferation and migration.Thiolutin,a BRCC36 inhibitor,effectively reduced the BRCC36/HMGCR interaction and suppressed HCC development.Conclusions:(1)HMGCR was a critical regulator in the interplay between ferroptosis and pyroptosis,and BRCC36 functioned as the DUB of HMGCR.(2)BRCC36 inhibited ferroptosis and promoted pyroptosis via deubiquitinating HMGCR.BRCC36 promoted the viability of proliferation and migration in HCC,and targeting BRCC36 inhibited cancer progression,implying that BRCC36 might provide a novel and promising therapeutic target for liver cancer treatment.更多还原