【作者】 陶海粟；

【导师】 黄志勇；

【作者基本信息】 华中科技大学 ， 外科学， 2022， 博士

【摘要】 第一部分:肝癌血管侵犯相关的lncRNAs调控网络和预后模型的构建目的:了解导致肝癌血管侵犯的危险因素对于评估患者复发风险和生存至关重要。本研究的目的是构建一个肝癌血管侵犯相关的lncRNA调控网络和预后模型。方法:本研究通过对癌症基因组图谱(TCGA)中具有血管侵犯信息的HCC患者的转录组测序数据进行差异分析,得到HCC血管侵犯相关差异基因。然后,应用加权相关网络分析的方法来筛选与HCC血管侵犯和预后相关的模块及核心基因。然后对关键模块中的基因进行分析来构建模型。同时,基于加权相关网络分析得到的关键基因,构建一个HCC血管侵犯相关的lncRNA-miRNA-mRNA ceRNA调控网络。结果:应用加权相关网络分析筛选出了与HCC血管侵犯和预后相关的模块及核心基因,基于这些核心基因我们构建了一个可以有效预测HCC患者总体生存期的lncRNA预后模型。在训练集、验证集、整个TCGA队列和同济队列中,高风险的患者风险评分均比低风险病例的总体生存期更短。此外,我们利用时间相关的受试者工作特征曲线来评估lncRNA预后模型的预测效能。结果显示,该lncRNA预后模型在这些数据集中均可以稳健地预测HCC病人预后。结论:本研究所建立的HCC血管侵犯相关的lncRNA预后模型可以有效地预测HCC病人的总体生存。并且初步探索了该lncRNA预后模型中的基因在肝细胞癌进展过程中的潜在作用机制及其临床应用价值。第二部分:关键分子BBOX1-AS1的表达与肝癌患者临床特征和预后的关系目的:从以上血管侵犯相关lncRNA预后模型中筛选出最关键的分子BBOX1-AS1。在包含83例本地肝细胞癌病人组织样本的队列中,检测BBOX1-AS1的表达水平。并结合病人的临床特征数据及随访资料,分析BBOX1-AS1的表达与肝癌病人的临床特征的相关性及其与病人预后的关系。方法:收集在武汉同济医院肝脏外科中心接受肝癌切除术的肝细胞癌病人的肿瘤组织及癌旁组织标本83例,所有标本均有同济医院病理科的明确诊断结果。从冰冻组织中提取RNA,并且进行实时定量PCR分析血管侵犯相关lncRNA预后模型中9个基因的表达量。通过对TCGA中肝细胞癌病人数据和83例同济队列中肝细胞癌病人数据进行分析,探讨BBOX1-AS1的表达情况与肝细胞癌病人的重要临床特征及预后之间的关系。结果:1.血管侵犯相关lncRNA预后模型中筛选出了关键分子BBOX1-AS1;2.和癌旁组织相比,BBOX1-AS1在肝癌组织中高表达,且BBOX1-AS1高表达的患者预后更差;3.BBOX1-AS1的表达水平与肿瘤分期和分级呈正相关,BBOX1-AS1是一个稳健的独立预后因子。结论:血管侵犯相关lncRNA BBOX1-AS1在肝癌组织中高表达,且BBOX1-AS1的高表达提示该病人肝癌肿瘤分期和分级更高、更容易复发,而且具有更差的预后。第三部分:BBOX1-AS1通过miR-361-3p/PHF8轴促进HCC进展和索拉非尼耐药目的:一些lncRNAs已被证实参与肝癌的进展和耐药。在上一步研究中,我们发现BBOX1-AS1在肝癌中高表达,并可能发挥致癌作用。需要进一步探索BBOX1-AS1的潜在功能和机制。方法:首先,证实BBOX1-AS1在HCC细胞系进展、自噬和耐药中的作用。然后通过FISH、RIP、双荧光素酶报告基因等一系列实验探讨其机制。最后,构建小鼠肿瘤模型和患者来源的类器官模型进行验证。结果:在本研究中,我们首次发现BBOX1-AS1可通过上调肝癌细胞的PHF8来促进肿瘤进展、自噬和耐药。在机制上,BBOX1-AS1通过吸附miR-361-3p来间接调控PHF8,从而影响HCC的进展和自噬。回复实验显示,PHF8是BBOX1-AS1和miR-361-3p在HCC中诱导的生物学效应的关键因子,提示BBOX1-AS1通过调控miR-361-3p/PHF8促进肿瘤进展和索拉非尼耐药。最后,我们在小鼠模型和类器官模型中证实了 BBOX1-AS 1敲减可以增强肝癌对索拉非尼的敏感性。结论:综上所述,BBOX1-AS1通过miR-361-3p/PHF8轴促进HCC进展和索拉非尼耐药。更多还原

【Abstract】 Part Ⅰ:Construction of a ceRNA Network and a Prognostic lncRNA Signature associated with Vascular Invasion in HepatocellularCarcinoma based on Weighted Gene Co-Expression Network Analysis Objective:Understanding risk factors for vascular invasion(Ⅵ)is crucial for assessing the risk of recurrence and overall prognosis of hepatocellular carcinoma(HCC).This study aimed to construct a prognostic long non-coding RNA(lncRNA)a ceRNA Network and signature associated with vascular invasion in HCC.Methods:Differentially expressed genes(DEGs)of HCC patients associated with VI were identified by analyzing data from TCGA.Weighted gene co-expression network analysis(WGCNA)was used to identify associations between gene expression modules and clinical features.A Ⅵ-related prognostic lncRNA signature was then stablished using univariate,LASSO and multivariate Cox proportional hazards regression analyses.Based on the hub modules identified by the WGCNA,we constructed a Ⅵ-related lncRNA-miRNA-mRNA ceRNA network and screened hub lncRNAs for further research.Results:The key module related to VI and OS was identified using WGCNA,after which a prognostic model consisting of nine lncRNAs was established,and verified using timedependent receiver operating characteristic(ROC)curve analysis.The results showed that the lncRNA prognostic signature could predict the prognosis of HCC patients in all these data sets,accurately.Conclusions:The Ⅵ-related lncRNA signature established in this study can be used to predict the clinical outcomes of HCC patients.In addition,we constructed a Ⅵ-related lncRNA-miRNA-mRNA ceRNA network which probably reflect all the interactions between these genes.Part Ⅱ:Relationship between expression of BBOX1-AS1 and clinical features and prognosis of HCC patientsObjective:To screen the most hub gene BBOX1-AS1 from the above vascular invasionrelated lncRNA prognostic model.The expression level of BBOX1-AS 1 was detected in the samples of 83 patients with HCC,and the correlation between the expression level of BBOX1-AS1 and the clinical characteristics of HCC patients and the prognosis of patients was analyzed based on the clinical characteristics and follow-up data of patients.Methods:83 tumor and paracancer tissue specimens of HCC patients who underwent tumor resection were collected in the Liver Surgery Center of Tongji Hospital,Wuhan.All the specimens were confirmed by the pathology department of Tongji Hospital.RNA was extracted from frozen tissues and the expression levels of 9 genes in the vascular invasionrelated lncRNA prognostic model were analyzed by qRT PCR.The relationship between BBOX1-AS1 expression and important clinical features and prognosis of hepatocellular carcinoma patients was investigated by analyzing the data of TCGA and 83 HCC patients in tongji cohort.Results:1.The hub gene BBOX1-AS1 was screened from the vascular invasion-related lncRNA prognostic model;2.BBOX1-AS1 is highly expressed in liver cancer tissues,and the high expression of BBOX1-AS1 suggests poor prognosis of HCC patients;3.The expression level of BBOX1-AS1 was positively correlated with tumor stage and grade,and BBOX1-AS1 was a robust independent prognostic factor.Conclusions:Vascular invasion-related lncRNA BBOX1-AS 1 is highly expressed in liver cancer tissues,and the high expression of BBOX1-AS1 suggests higher stage and grade of tumor and worse prognosis.Part Ⅲ:Oncogenic lncRNA BBOX1-AS1 promotes PHF8-mediated autophagy and elicits sorafenib resistance in hepatocellular carcinoma Objective:Some long non-coding RNAs(lncRNAs)have been documented to be involved in cancer progression and anticancer drug resistance in hepatocellular carcinoma(HCC).Thus,approaches designed to target these genes may facilitate the development of promising strategies for treating HCC.Previously,we showed that lncRNA BBOX1-AS1 was highly expressed and might play an oncogenic role in HCC.However,the potential functions and mechanisms through which BBOX1-AS1 regulates HCC progression and drug resistance remain unclear.Methods:Firstly,the function of BBOX1-AS 1 on the progression,autophagy and drug resistance of HCC was demonstrated HCC cell lines.Then,the mechanism was explored by a series of experiment including FISH,RIP and dual-luciferase reporter assays.Results:In this study,we for the first time revealed that BBOX1-AS 1 could promote tumor progression,autophagy and drug resistance by upregulating PHF8 in HCC cells.Mechanistically,BBOX1-AS1 enhanced the stability of PHF8 mRNA by targeting the PHF 8 inhibitor miR-361-3p to regulate tumor progression and autophagy in HCC.The functional rescue experiments showed that PHF8 acted as a key factor in regulating the biological effects induced by BBOX1-AS1 and miR-361-3p in HCC,indicating that BBOX1-AS1 promotes tumor progression and sorafenib resistance by regulating miR-361-3p/PHF8.Finally,mouse tumor models and patient-derived organoid models were established to further confirm these findings.Conclusions:Taken together,the results demonstrate that BBOX1-AS1 promotes HCC progression and sorafenib resistance via the miR-361-3p/PHF8 axis.更多还原