PPARA参与肝细胞癌发生铁死亡预防疾病恶化的初步研究

Preliminary study of PPARA regulate the ferroptosis of hepatocellular carcinoma to prevent disease deterioration

本研究通过在线公共数据库与细胞实验分析过氧化物酶体增殖物激活受体a(peroxisome proliferator-activated receptor a,PPARA)在肝细胞癌中的表达情况、基因功能及对肝细胞癌(hepatocellular carcinoma,HCC)患者疾病恶化的影响,探讨PPARA是否参与肝细胞癌铁死亡的过程。采用实验性研究,基于生物信息学的数据分析及细胞实验,2022年1至8月在我院基础医学实验室进行相关细胞实验。利用癌症基因组数据(The Cancer Genome Atlas,TCGA)和基因表达数集(Gene Expression Omnibus,GEO)数据库分析PPARA在肝细胞癌中表达水平及与患者临床病理特征相关性。通过人类蛋白免疫组化表达数据库(The Human Protein Atlas,HPA)研究PPARA在HCC肿瘤组织及正常组织中蛋白表达情况。基于基因、蛋白质相互作用关系检索工具(Search Tool for the Retrival of Interacting Genes/Protein,STRING)数据库构建PPARA与铁死亡关键因子的蛋白质相互作用(protein-protein interaction,PPI)网络,同时用基因表达谱数据动态分析(Gene Expression Profiling Interactive Analysis,GEPIA)分析PPARA与关键基因谷氨酸半胱氨酸连接酶催化亚单位(Glutamate-Cysteine Ligase Catalytic Subunit,GCLC)的相关性。通过免疫印迹(Western Blot,WB)检测HCC细胞株SK-HEP-1、SMMC-7721、MHCC-97H、BEL-7402及正常肝细胞L02中PPARA的表达水平,观察用铁死亡诱导剂Erastin处理48 h后PPARA表达量的变化。GEPIA数据库中各组间PPARA表达量间比较使用单因素方差分析方法。GSE25097与GSE112790数据集中PPARA的表达量比较采用秩和检验。生存分析使用时序检验方法进行统计。比较不同临床、病理特征间PPARA表达量间的差异利用克鲁斯卡尔-沃利斯检验。利用斯皮尔曼相关系数的方法比较GCLC与PPARA表达量之间的相关性。利用配对T检验对PPARA在细胞系中表达量进行比较。结果显示,HCC组织中PPARA的RNA水平和蛋白表达水平均低于正常组织(P<0.05)。PPARA表达水平与临床病理分级和预后有关(P<0.05)。STRING数据库构建的PPI提示PPARA与铁死亡关键因子NFE2样bZIP转录因子2(NFE2 like bZIP transcription factor 2,NFE2L2)、血红素加氧酶1(heme oxygenase 1,HMOX1)、肿瘤蛋白P53(tumor protein P53,TP53)、GCLC、二肽基肽酶4(dipeptidyl peptidase 4,DPP4)、柠檬酸合成酶(citrate synthase,CS)、花生四烯酸15脂氧合酶(arachidonate 15-lipoxygenase,ALOX15)、酰基CoA合成酶长链家族成员4(Acyl-CoA synthetase long chain family member 4,ACSL4)等存在相互作用。进一步研究表明,GEPIA数据库结果显示PPARA与GCLC的表达呈正相关(R=0.6,P<0.05)。用铁死亡诱导剂Erastin处理MHCC-97H和SK-HEP-1细胞48 h后,通过WB检测发现PPARA表达量均升高。综上,PPARA在HCC低表达,表达量越低则患者生存期越短。PPARA与GCLC相互作用,从而共同参与调控HCC铁死亡过程。

To explore whether PPARA is involved in the process of ferroptosis in hepatoma cells,peroxisome proliferator activated receptor(PPARA)was comprehensively analyzed in hepatocellular carcinoma(HCC)through public database and experimental data,including the expression,the functions and the potential roles of tumor progression.The research design is experimental research,data analysis based on bioinformatics and cell experiment.From January 2022 to August 2022,relevant cell experiments were conducted in the Basic Medical Laboratory of the General Hospital of the Southern Theatre of the Chinese People′s Liberation Army.The expression and the correlation with clinicopathologic features of PPARA in HCC were analyzed by The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)databases.To study the protein expression of PPARA in HCC and normal tissues through the Human Protein Atlas(HPA).The protein-protein interaction(PPI)network between PPARA and the core factor of ferroptosis was constructed based on Search Tool for the Retrival of Interacting Genes/Protein(STRING)database,then,the correlation between PPARA and the core gene Glutamate-cysteine Ligase Catalytic Subunit(GCLC)was analyzed by Gene Expression Profiling Interactive Analysis(GEPIA).Assessed the expression of PPARA in HCC cell lines SK-HEP-1,SMMC-7721,MHCC-97H,BEL-7402 and normal liver cell L02 by Western Blot(WB)and the changes of PPARA expression after 48h treatment with ferroptosis inducer Erastin were observed.Single factor analysis of variance was used to compare the expression of PPARA between groups in GEPIA database.The expression of PPARA in GSE25097 and GSE112790 data was compared by rank sum test.Survival analysis was performed using time series test method.The difference of PPARA expression between clinical and pathological features was compared using the Kruskal-Wallis test.The correlation between the expression of GCLC and PPARA was compared by the method of Spearman correlation.The expression of PPARA in cell lines was compared by paired T test.The results showed that the RNA and protein expression of PPARA in HCC was lower than that in normal tissues(P<0.05).PPARA alterations were correlated with patient clinicopathological features and prognosis(P<0.05).The PPI constructed by STRING database suggests that PPARA interact with the key factors of ferroptosis,such as NFE2 like bZIP transcription factor 2(NFE2L2),Heme Oxygenase 1(HMOX1),Tumor Protein P53(TP53),GCLC,Dipeptidyl Peptidase 4(DPP4),Citrate Synthase(CS),Arachidonate 15-Lipoxygenase(ALOX15)and Acyl-CoA Synthetase Long Chain Family Member 4(ACSL4).Furthermore,the PPARA was significantly associated with GCLC validated via GEPIA database(R=0.6,P<0.05).The expression of PPARA increased after treatment with ferroptosis inducer Erastin for 48 h by WB.In conclusion,the expression of PPARA is lower in HCC with a poor prognosis.PPARA interacts with GCLC in regulating ferroptosis in HCC.