【作者】 刘波；

【导师】 杜成友；

【作者基本信息】 重庆医科大学 ， 外科学（专业学位）， 2024， 博士

【摘要】 第一部分基于长非编码RNA的肝癌预后模型构建目的:肝细胞癌(HCC)是目前威胁人类健康的一种预后较差的恶性肿瘤。N6-甲基腺苷(m6A)甲基化修饰参与多种肿瘤的发生和发展。长非编码RNA(lnc RNA)的异常表达与肝癌的发生发展及肝癌患者的预后密切相关。本部分旨在筛选与肝癌预后相关且与m6A修饰基因的表达相关的lnc RNA并构建肝癌的预后预测模型,探索模型对肝癌患者生存预后的预测价值。方法:从癌症基因组图谱(TCGA)数据库中获取肝癌患者的转录组数据及临床资料。采用Pearson相关性分析筛选与m6A修饰基因的表达具有相关性的lnc RNA,进一步采用Cox回归分析筛选与肝癌预后相关的lnc RNA。按照7:3的比例将肝癌患者随机划分为训练集和测试集。在训练集中,利用最小绝对收缩和选择算子(LASSO)和Cox回归分析筛选最佳lnc RNA用于构建肝癌预后模型,并利用模型计算肝癌患者的风险评分。通过Kaplan-Meier生存分析和时间依赖的受试者工作特征(ROC)曲线评估模型预测肝癌患者预后的能力,并在测试集中进行验证。随后探索预后模型与肝癌临床特征之间的关系,分析比较不同风险评分的肝癌患者的肿瘤免疫微环境的差异以及不同风险评分的肝癌患者对免疫治疗的反应。结果:采用Pearson相关性分析,从TCGA的HCC数据集中筛选出491个与m6A修饰基因的表达显著相关的lnc RNA。进一步筛选发现其中65个lnc RNA与HCC患者的预后显著相关。在训练集中,通过结合LASSO和Cox回归方法,筛选出10个最佳lnc RNA用于构建HCC预后预测模型。通过模型为每位患者计算风险评分,并通过中位风险评分将肝癌患者划分为高风险组和低风险组。Kaplan-Meier生存分析结果表明,高风险组HCC患者的生存时间明显短于低风险组患者。ROC分析表明模型在预测HCC患者总体生存(OS)方面表现良好。在验证集中进行验证也得到了一致的结果。单因素及多因素Cox回归分析显示模型风险评分和肿瘤T分期是HCC患者预后的独立危险因素。另外,高风险组患者的免疫检查点基因表达水平显著高于低风险组。低风险组患者对免疫治疗呈现出相对较好的反应。结论:基于lnc RNA构建的肝癌预后模型在预测肝癌患者的预后方面表现良好,有助于在临床上对肝癌患者进行分层管理。模型内的lnc RNA可能是肝癌治疗的潜在靶点。第二部分长非编码RNA EMC3-AS1在肝癌内的表达及作为肝癌预后和诊断标志物的价值目的:本部分旨在进一步探索在上一部分中构建的预后模型内纳入的lnc RNA之一EMC3-AS1在肝癌中的表达,并评价其作为肝癌预后及诊断标志物的价值。方法:收集临床肝癌标本,通过定量PCR分析EMC3-AS1在肝癌组织及癌旁组织中的表达差异,并在TCGA数据库及基因表达数据库(GEO)中的3个肝癌数据集中验证EMC3-AS1的表达。评价EMC3-AS1预测肝癌预后的能力和区分肝癌组织与癌旁组织的能力。探索EMC3-AS1的表达与肝癌患者临床特征之间的关系。比较EMC3-AS1高表达组和低表达组的肝癌患者之间在肿瘤免疫微环境及对免疫治疗的反应等方面的差异。结果:定量PCR检测结果表明,EMC3-AS1在肝癌组织中的表达水平显著高于癌旁组织,在TCGA数据库及3个GEO肝癌数据集中得到了一致的结果。ROC结果显示EMC3-AS1区分肝癌及癌旁组织的能力表现良好。EMC3-AS1的表达与肝癌患者的较短的总体生存和无疾病生存相关。单因素及多因素Cox回归分析显示EMC3-AS1的表达(HR,1.24;95%CI,1.07-1.45;P=0.006)和肿瘤T分期(HR,2.30;95%CI,1.58-3.37;P<0.001)是影响肝癌预后的独立危险因素。肿瘤免疫微环境及免疫治疗反应分析结果显示,EMC3-AS1表达较低的肝癌患者更有可能在免疫治疗中获益。结论:EMC3-AS1在肝癌组织中高表达,EMC3-AS1的表达是影响肝癌预后的独立危险因素。EMC3-AS1可能是肝癌潜在的预后标志物和诊断标志物。第三部分长非编码RNA EMC3-AS1对肝癌细胞增殖和迁移的影响与机制目的:本部分旨在探讨EMC3-AS1对肝癌细胞增殖和迁移等生物学行为的影响,初步探讨其影响肝癌细胞生物学行为的机制。方法:采用si RNA敲低肝癌细胞Sk-Hep-1、Huh-7和Hep G2中EMC3-AS1的表达,通过MTT实验和细胞克隆形成实验观察在敲低EMC3-AS1的表达之后肝癌细胞的增殖能力的变化,通过划痕实验和transwell实验观察在敲低EMC3-AS1的表达之后肝癌细胞迁移能力的变化。采用核质分离实验检测EMC3-AS1在细胞内的定位。筛选与EMC3-AS1潜在结合的mi RNA以及mi RNA的下游靶m RNA。采用双荧光素酶报告实验,验证EMC3-AS1与mi RNA的结合关系,并通过过表达mi R-125b-5p或mi R-129-5p后检测下游m RNA的表达变化。结果:在肝癌细胞Sk-Hep-1、Huh-7和Hep G2中敲低EMC3-AS1的表达之后,MTT实验和平板细胞克隆形成实验结果显示肝癌细胞Sk-Hep-1、Huh-7和Hep G2的增殖能力明显受抑。划痕实验和transwell结果表明,肝癌细胞Sk-Hep-1、Huh-7和Hep G2的迁移能力在敲低EMC3-AS1的表达之后明显降低。敲低EMC3-AS1的表达之后,mi R-125b-5p和mi R-129-5p的表达显著上调。双荧光素酶实验证实EMC3-AS1与mi R-125b-5p或mi R-129-5p存在直接结合关系。在分别过表达mi R-125b-5p和mi R-129-5p之后,我们发现mi R-125b-5p的靶m RNA包括KIF18B、IGSF3和SLC38A1的表达显著降低;mi R-129-5p的靶m RNA包括ABCC5和HOXB9的表达显著降低。在抑制肝癌细胞mi R-125b-5p或mi R-129-5p的表达之后,可以逆转敲低EMC3-AS1导致的肝癌增殖和迁移能力的抑制作用。结论:敲低肝癌细胞内EMC3-AS1的表达后,肝癌细胞的增殖能力及迁移能力明显受到抑制。EMC3-AS1可能通过“海绵”吸附mi R-125b-5p和mi R-129-5p,促进肝癌细胞增殖和迁移。更多还原

【Abstract】 SECTION ONE:CONSTRUCTION OF A PROGNOSTIC MODEL FOR HEPATOCELLULAR CARCINOMA BASED ON LONG NON-CODING RNASObjective:Hepatocellular carcinoma(HCC)is a malignancy with poor prognosis that poses a significant threat to human health.Increasing evidence suggests that N6-methyladenosine(m6A)methylation modification is involved in the development and progression of various tumors.The abnormal expression of long non-coding RNAs(lnc RNAs)is closely associated with the development of HCC and the prognosis of HCC patients.The aim of this section is to screen for lnc RNAs that are related to the prognosis of HCC and correlated with the expression of m6A-modified genes,and to construct a prognostic prediction model for HCC.Addittionally,the predictive value of the model in the survival prognosis of patients with HCC was expolored.Methods:Transcriptomic data and clinical information of HCC patients were obtained from The Cancer Genome Atlas(TCGA)database.Pearson correlation analysis was utilized to screen out lnc RNAs correlated with the expression of m6A modification genes.Cox regression analysis was used to identify lnc RNAs associated with HCC prognosis.HCC patients were randomly divided into a training set and a test set in a 7:3ratio.In the training set,the least absolute shrinkage and selection operator(LASSO)regression and Cox regression analyses were employed to select the optimal lnc RNAs for constructing a HCC prognostic model,and risk scores were computed for each patient.The predictive capacity of the model regarding HCC patient prognosis was assessed using Kaplan-Meier survival analysis and time-dependent receiver operating characteristic(ROC)curves,and was validated in the test set.Additionally,the relationship between the prognostic model and clinical characteristics of HCC was explored,and the differences in tumor immune microenvironment among HCC patients with different risk scores,as well as their responses to immunotherapy based on these risk scores,were analyzed and compared.Results:Using Pearson correlation analysis,491 lnc RNAs significantly correlated with the expression of m6A modification genes were identified.Further screening revealed that 65 of these lnc RNAs were significantly associated with the prognosis of HCC patients.In the training set,a combination of LASSO and Cox regression methods identified 10optimal lnc RNAs for constructing the HCC prognostic prediction model.Risk scores of HCC patients were calculated using the model,and the patients were divided into high-risk and low-risk groups based on the median risk score.Kaplan-Meier survival analysis indicated that the overall survival(OS)of high-risk group was significantly shorter than that of the low-risk group.ROC analysis demonstrated that the model performed well in predicting OS for HCC patients.Consistent results were obtained in the test set.Univariate and multivariate Cox regression analyses demonstrated that risk scores and T stage were independent risk factors for the prognosis of HCC patients.Furthermore,the expression levels of immune checkpoint genes were significantly higher in the high-risk group compared to the low-risk group,and the low-risk group exhibiting a better response to immunotherapy.Conclusions:The prognostic model,constructed from m6A-related long non-coding RNAs(lnc RNAs),exhibits good performance in predicting outcomes for HCC patients and may facilitate stratified management in clinical practice.Furthermore,these lnc RNAs involved in the prognostic model could potentially serve as therapeutic targets in HCC treatment.SECTION TWO:EXPRESSION OF LNCRNA EMC3-AS1 IN HEPATOCELLULAR CARCINOMA AND ITS POTENTIALAS A PROGNOSTIC AND DIAGNOSTIC BIOMARKERObjective:This section aims to further investigate the expression of EMC3-AS1,one of the long non-coding RNAs incorporated in the prognostic model constructed previously for HCC,and evaluate its potential as a prognostic and diagnostic biomarker for HCC.Methods:Clinical HCC specimens were collected,and quantitative PCR was used to analyze the expression differences of EMC3-AS1between HCC tissues and adjacent normal tissues.The expression of EMC3-AS1 in TCGA and three HCC datasets from Gene Expression Omnibus(GEO)was validated.The ability of EMC3-AS1 to predict HCC prognosis and distinguish between tumor tissues and adjacent normal tissues was assessed.Additionally,the relationship between the expression of EMC3-AS1 and the clinical characteristics of HCC patients was explored.The differences of tumor immune microenvironment and responses to immunotherapy between high-and low-expression EMC3-AS1 groups were compared.Results:Quantitative PCR results indicated that EMC3-AS1 was significantly upregulated in HCC tissues compared to adjacent normal tissues,which was consistent with the findings in TCGA and all three HCC datasets from GEO.EMC3-AS1 effectively distinguished between HCC tissues and adjacent normal tissues,demonstrating good performance in both the TCGA and GEO datasets,as well as in the current clinical cohort.The expression of EMC3-AS1 was associated with shorter OS and disease-free survival(DFS)in HCC patients.Univariate and multivariate Cox regression analyses revealed that EMC3-AS1 expression(HR,1.24;95%CI,1.07-1.45;P=0.006)and T stage(HR,2.30;95%CI,1.58-3.37;P<0.001)were independent risk factors affecting HCC prognosis.Analysis of the tumor immune microenvironment and response to immunotherapy indicated that HCC patients with low EMC3-AS1 expression were more likely to benefit from immunotherapy.Conclusions:EMC3-AS1 is overexpressed in HCC tissues and serves as an independent risk factor for HCC prognosis.It holds potential as a prognostic and diagnostic biomarker for HCC patients.SECTION THREE:IMPACT OF LNCRNA EMC3-AS1 ON PROLIFERATION AND MIGRATION OF LIVER CANCER CELLS AND THE UNDERLYING MECHANISMSObjective:This section aims to investigate the effects of EMC3-AS1on biological behaviors such as proliferation and migration of liver cancer cells,and to preliminary explore the mechanisms by which it influences these cellular activities.Methods:Small interfering RNA(si RNA)was employed to knock down the expression of EMC3-AS1 in liver cancer cell lines Sk-Hep-1,Huh-7,and Hep G2.The changes in cell proliferation following the knockdown of EMC3-AS1 in liver cancer cell were assessed using MTT assays and colony formation assays.The migration capabilities of the liver cancer cells were evaluated using wound healing assays and transwell migration assays after knocking down the expression of EMC3-AS1.Nuclear-cytoplasmic separation experiments were conducted to determine the cellular localization of EMC3-AS1.We identified the potential mi RNAs that interact with EMC3-AS1 and their downstream target m RNAs.The binding relationship between EMC3-AS1 and the identified mi RNAs were validated through dual-luciferase reporter assays.Results:After knocking down EMC3-AS1 expression in liver cancer cell lines Sk-Hep-1,Huh-7,and Hep G2,MTT and colony formation assays revealed a significant inhibition of cell proliferation.Wound healing and transwell assays indicated a significant reduction in the migration ability of these liver cancer cells after EMC3-AS1 knockdown.The expression levels of mi R-125b-5p and mi R-129-5p were significantly upregulated upon knocking down EMC3-AS1 in liver cancer cells.Dual-luciferase assays confirmed a direct binding relationship between EMC3-AS1 and mi R-125b-5p,as well as mi R-129-5p.Furthermore,after transfection with mi R-125b-5p mimics or mi R-129-5p mimics,we observed a significant reduction in the expression of mi R-125b-5p target m RNAs,including KIF18B,IGSF3 and SLC38A1,as well as a decrease in the expression of mi R-129-5p target m RNAs ABCC5 and HOXB9.Inhibition of mi R-125b-5p or mi R-129-5p expression could reverse the suppressive effects of EMC3-AS1 knockdown on proliferation and migration of liver cancer cells.Conclusions:The knockdown of EMC3-AS1 expression in liver cancer cells significantly inhibits their proliferation and migration abilities.EMC3-AS1 may act as a“sponge”for mi R-125b-5p and mi R-129-5p,thereby promoting proliferation and migration of liver cancer cells.更多还原