【作者】 王正；

【导师】 马立宪；

【作者基本信息】 山东大学 ， 内科学（传染病）， 2021， 博士

【摘要】 肝细胞癌(hepatocellular carcinoma、HCC)是全球最多见的恶性肿瘤之一,恶性水平高,发展迅猛。我国是HCC的高发区,目前占世界的55%。由于乙型肝炎病毒的长期感染,我国多半病人在慢性肝炎,肝硬化后发展成HCC。HCC具备起病隐匿、早期诊断困难、预后差、进展快等特点。HCC的死亡率是肿瘤的第二位。因此,探求一种新的有效的生物标志物对HCC的诊断、预后和治疗具有重大意义。层出不穷的证据表明免疫系统在癌症发生和发展过程中起决定性作用。免疫细胞不仅调节机体的抗肿瘤免疫,并且也对肿瘤细胞增殖和转移等病理变化起到重要的调控作用。免疫治疗近年来越发受到重视,特别是随着CTLA-4(细胞毒T淋巴细胞相关抗原4)和PD-1(程序性死亡受体1)抗体取得良好的临床效果。例如,作为抗PD-1单克隆抗体Nivolumab(纳武利尤单抗)可以通过干扰信号通路阻断PD-1,恢复机体的抗癌免疫反应。免疫治疗可为HCC提供一种安全、有效、有前途的治疗方式。但肝脏的免疫耐受性和肿瘤本身的异质性仍然是主要问题。探求新的影响预后以及免疫治疗的免疫靶基因具有特别重要的价值。国内外对免疫相关基因的研究还很局限。尽管HCC研究中测序技术及芯片技术不断提高,仍然有大量HCC中差异表达免疫相关基因的功能调控分子机制不清楚。到目前为止,仍没有筛选到可以用于临床的诊断、预后或治疗靶标的免疫相关基因。DCK(Deoxycytidine kinase,脱氧胞苷激酶)属于DCK/DGK家族,是几种脱氧核苷及其类似物磷酸化所必需的。DCK也是脱氧核糖核苷挽救的关键酶,是脱氧核糖核酸修复过程中的关键酶,对维持正常的DNA代谢具有重要意义。目前研究表明DCK缺乏与对抗病毒和抗癌化疗药物的耐药性有关。同时有研究报道,DCK在乳腺癌、宫颈癌、食管癌等肿瘤的发生发展中起到重要作用。我们通过生物信息分析学分析发现DCK在HCC中高表达,并且与HCC患者生存相关,DCK的异常高表达提示患者预后不良。然而,DCK在HCC中的具体作用机制尚未报道,有待于进一步探讨。目前,关于免疫相关基因在HCC中的研究尚处于起步阶段,探索和发现新的免疫相关基因及其特异性调控机制,可为研究HCC的发病机制提供新的理论依据,同时为发现新的治疗靶点提供理论基础。本文以此为出发点,研究内容如下:一,以TCGA数据库(The Cancer Genome Atlas,癌症和肿瘤基因组图谱)和 ImmPort 数据库(The Immunology Database and Analysis Portal,免疫学数据库和分析门户)为基础,利用生物信息学分析筛选出HCC组织及癌旁组织间的差异表达免疫相关基因,进一步挖掘对HCC患者具备预后价值的免疫相关基因。二,多个公开数据库明确肝癌中DCK的表达情况,分析DCK和HCC免疫微环境的关系,检测乙肝相关肝癌及癌旁组织样本中DCK表达,并分析DCK与临床病理参数的关系。三,通过沉默人肝癌细胞系中DCK表达,研究DCK对肝癌细胞系各种恶性生物学行为的影响;通过裸鼠成瘤实验评价DCK对肝癌细胞HepG2体内成瘤能力的影响;初步探讨DCK调控HCC发生进展的分子机制。第一部分 肝细胞癌候选免疫相关基因的筛选目的:以TCGA数据库和ImmPort数据库为基础,利用生物信息学分析筛选出HCC组织及癌旁组织间的差异表达免疫相关基因,进一步挖掘对HCC患者具有预后价值的免疫相关基因,并确定在HCC发生发展过程中起关键作用的免疫相关基因。方法:1.应用TCGA数据库、ImmPort数据库筛选HCC组织中差异表达的免疫相关基因。下载TCGA数据库中的HCC患者的基因表达数据及临床数据,在ImmPort数据库中下载1811个免疫相关基因。在R软件中用Wilcoxon检验法检测HCC组织和癌旁组织中差异表达的免疫相关基因,评估差异表达免疫相关基因的潜在生物学功能。2.筛选与生存相关的差异表达免疫相关基因。只对随访时间少于2000天的HCC患者进行生存分析。在R软件中分别利用单因素Cox分析和Kaplan-Meier生存分析方法查找生存相关的差异表达免疫相关基因,P值小于0.01作为筛选条件,使用两种方法查找后,保留共同存在的有预后价值的差异表达免疫相关基因。3.独立预后因子的筛选。将上一步得到的每一个基因和临床数据(年龄、分级、病理分期、T分期等)一起进行多因素Cox分析,P值小于0.01作为筛选条件,筛选出独立预后免疫相关基因。4.预测分析调控HCC独立预后免疫相关基因的转录因子。为了研究免疫相关基因的调控机制,我们提取了顺反组癌症数据库(Cistromecancer)里的 318 个转录因子(Transcriptionfactors,TFs)进行后续研究。利用TCGA中数据找出差异表达的转录因子,对它们进行生存分析。然后使用R中的cor.test函数得到生存相关的转录因子与独立预后免疫相关基因的相关性。过滤标准为相关系数大于0.5,P值小于0.05。Cytoscape软件用于构建和可视化调控网络。结果:1.HCC组织中差异表达的免疫相关基因结果。从TCGA数据库中下载374例HCC和50例癌旁组织预处理的RNA-Seq-FPKM数据进行下一步分析。HCC组织与癌旁组织相比共有329个免疫相关基因(267个上调,62个下调)差异表达。通过GO和KEGG分析进一步分析了329个差异表达的免疫相关基因的生物学功能。2.筛选与生存相关的差异表达免疫相关基因结果。我们利用差异分析得到的329个差异表达的免疫相关基因进行生存分析。通过原始数据发现随访时间少于2000天的HCC患者共337例。应用单因素Cox分析对337例HCC组织中329个差异免疫相关基因的表达进行检测,共发现64个生存相关的免疫相关基因。采用Kaplan-Meier生存分析方法得到58个生存相关的免疫相关基因。应用韦恩图找到32个共同存在的生存相关的免疫相关基因,均为高风险基因。3.独立预后免疫相关基因筛选结果。将上述得到的32个免疫相关基因进行独立预后分析,得到12个免疫相关基因可以作为独立预后基因。其中,DCK在多种恶性肿瘤中异常表达并参与肿瘤的起始和进展。然而DCK在HCC中的具体作用机制尚未阐明,有待于进一步探讨。4.转录因子与独立预后免疫相关基因的调控网络构建。HCC组织中与癌旁组织相比,共有117个转录因子(108个上调,9个下调)差异表达。应用单因素Cox分析和Kaplan-Meier生存分析方法得到27个生存相关的转录因子。根据过滤标准,共鉴定出25个生存相关的转录因子和9个独立预后免疫相关基因,以建立网络。其中,HCFC1调节了大多数独立预后免疫相关基因。我们预测DCK最重要的上游转录因子是HCFC1。结论:1.通过对TCGA和ImmPort数据库分析,得到HCC 12个差异表达的免疫相关基因与预后密切相关并且可以独立预测预后。其中DCK在HCC中的生物学作用尚未明确,可能是HCC的关键免疫相关基因。2.预测出25个转录因子可以调控一个或多个免疫相关基因,其中HCFC1在HCC中有重要的调控作用,初步明确了 HCFC1是DCK最重要的上游调控转录因子。第二部分 DCK在肝癌中的表达及与乙肝相关肝癌患者病理参数的相关分析目的:多个公开数据库明确肝癌中DCK的表达情况。探讨DCK和HCC免疫微环境的关系,预测HCC中DCK调控的相关通路。检测乙肝相关肝癌组织中DCK的表达,研究DCK表达与乙肝相关肝癌患者临床病理参数的关系。检测人不同的肝癌细胞系(HepG2,SKHEP1)和正常肝细胞HL-7702中DCK mRNA和蛋白的表达水平。方法:1.通过oncomine数据库、GEPIA网站和ICGC数据库,查询DCK在肝癌组织及对应正常组织中的表达情况。2.利用 TIMER 网站(Tumor Immune Estimation Resource,肿瘤免疫评估资源)调查TCGAHCC患者DCK与HCC免疫微环境的关系;通过GSEA方法预测HCC中DCK调控的相关通路。3.通过实时荧光定量PCR(qRT-PCR)检测53例乙肝相关肝癌肿瘤组织及癌旁组织样本中DCK表达量,分析DCK与临床病理参数的关系。4.实时荧光定量PCR和Western blot分别检测了人不同的肝癌细胞系(HepG2,SKHEP1)和正常肝细胞HL-7702中DCK的表达水平。结果:1.多个数据库结果均显示,DCK在肝癌组织较正常组织高表达。2.DCK表达与HCC免疫微环境中CD4+T细胞、中性粒细胞、树突状细胞、B细胞、巨噬细胞和CD8+T细胞均正相关。DCK可用于评估肿瘤组织中免疫细胞浸润的水平。GSEA显示DCK可以正调控多种通路进而影响HCC的发生发展,包括WNT信号通路,ERBB信号通路等。3.实时荧光定量PCR检测显示,乙肝相关肝癌患者肝癌组织中DCK表达水平较癌旁组织显著升高。DCK的表达与年龄、性别无关,但与肿瘤大小、Edmondson分级、微血管侵犯相关。4.HepG2,SKHEP1中DCK的mRNA和蛋白表达水平均高于正常肝细胞HL-7702。结论:DCK在肝癌组织中呈高表达。DCK表达水平与HCC免疫微环境中免疫细胞正相关,提示DCK可以反映HCC免疫微环境的状况。DCK在乙肝相关肝癌患者癌组织中高表达,且与多种临床病理参数相关。HepG2,SKHEP1中DCK mRNA和蛋白均高表达,可用于后续实验。第三部分 DCK对肝癌细胞恶性生物学行为的影响目的:通过沉默肝癌细胞HepG2,SKHEP1中的DCK表达,检测DCK对肝癌细胞增殖、凋亡、侵袭等恶性生物学行为的影响,然后经过裸鼠成瘤实验进一步验证;初步探讨DCK促进HCC发生进展的分子机制。方法:应用siRNA技术(small interfering RNA,小干扰RNA)沉默DCK在肝癌细胞系中的表达,采用qRT-PCR和Western blot筛选出干扰效率最高的序列。利用CCK-8增殖实验、细胞划痕实验、Transwell侵袭实验、流式细胞技术检验DCK基因低表达后肝癌细胞增殖、迁移、侵袭和凋亡能力。裸鼠皮下注射DCK沉默组(siRNA-DCK组)和阴性对照组siRNA(NC组)的肝癌细胞HepG2。4周后处死裸鼠,剥离肿瘤并称量。利用Western blot、细胞免疫荧光检测了肝癌细胞中干扰DCK后,对WNT信号通路主要蛋白水平(Wnt-1,β-catenin,c-myc)的影响。结果:成功筛选出1条siRNA用于后续实验。CCK-8实验发现沉默表达DCK的肝癌细胞系HepG2和SKHEP1的增殖能力显著减弱。细胞划痕实验及Transwell侵袭实验显示沉默表达DCK的肝癌细胞HepG2和SKHEP1细胞的迁移和侵袭能力显著减弱。流式分析显示DCK下调可促进肝癌细胞的凋亡。动物实验结果表明,DCK沉默组肿瘤生长速度缓慢,质量及体积明显减小。抑制DCK表达后WNT信号通路主要蛋白表达降低,这提示DCK异常活化WNT信号通路,使关键信号分子Wnt-1,β-catenin,c-myc表达增加,促进肝癌的恶性进程。结论:体外实验中,沉默DCK基因抑制肝癌细胞HepG2和SKHEP1的增殖、迁移和侵袭能力、促进肝癌细胞的凋亡。动物实验表明,沉默DCK对裸鼠成瘤能力具有显著的抑制作用。DCK可能通过激活WNT信号通路影响肝癌的恶性生物学行为。DCK有望作为HCC患者治疗新的靶标。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide.China has a high incidence of HCC,accounting for 55%of the world’s incidence.Due to the long-term infection of hepatitis B virus,most patient in China developed into HCC after chronic hepatitis and cirrhosis.HCC is characterized by insidious onset,difficult early diagnosis,poor prognosis and rapid progression.The mortality rate ranks second in tumors,so it is crucial to find a new and effective biomarker for the diagnosis,prognosis and treatment of HCC.There is growing evidence that the immune system plays a decisive role in the development and progression of cancer.Immune cells not only regulate the body’s anti-tumor immunity,but also play an important role in regulating the proliferation and metastasis of tumor cells.Immunotherapy has received more and more attention in recent years,especially with the clinical effect of CTLA-4 and PD-1 antibodies.For example,as an anti-PD-1 monoclonal antibody,Nivolumab can block PD-1 by interfering with signal pathways and restore the body’s anti-cancer immune response.In HCC,Immunotherapy can provide a safe,effective and promising treatment for HCC.However,the liver’s immune tolerance and the heterogeneity of the tumor itself are still the main problems.It is of great value to find new immune target genes that affect prognosis and immunotherapy.The research on immune-related genes is still limited at home and abroad.Despite the continuous improvement of sequencing technology and microarray technology in HCC research,there are still a large number of differentially expressed immune-related genes in HCC,and the molecular mechanism of functional regulation is unclear.So far,no immune-related genes have been screened for clinical diagnosis,prognosis or therapeutic targets.DCK(deoxycytidine kinase)belongs to the DCK/DGK family and is essential for the phosphorylation of several deoxynucleotides and their analogues.DCK is also a key enzyme in DNA repair,which plays a vital role in keeping normal DNA metabolism.Current studies have shown that the lack of DCK is related to the resistance of anti-virus and anti-cancer chemotherapy drugs.At the same time,studies have reported that DCK plays an important role in the occurrence and development of breast cancer,cervical cancer,esophageal cancer and other tumors.Through bioinformatics analysis,we noted that DCK was highly expressed in HCC,which was associated with the survival of HCC patients.The abnormal high expression of DCK indicated poor prognosis.However,the specific mechanism of DCK in HCC has not been reported,and needs to be further explored.Exploring and discovering new immune-related genes and its specific regulatory mechanism can provide a new theoretical basis for the study of pathogenesis of HCC,and provide a theoretical basis for the discovery of new therapeutic targets.This paper takes this as the starting point,and the research contents are as follows:Part I,Based on the TCGA database and the ImmPort database,differentially expressed immune-related genes between HCC tissues and adjacent tissue were screened by using bioinformatics analysis.We further excavated immune-related genes that have prognostic value for patients with HCC,and identified immune-related genes that play a key role in the development of HCC.Part II,several open databases were established to identify the expression of DCK in HCC.We focused on the relationship between DCK and immune microenvironment.Then we detected the expression of DCK in HBV-related HCC and adjacent tissues,and investigated the relevance between DCK and clinicopathological characteristics.Part Ⅲ,By silencing the expression of DCK in HepG2 and SKHEP1,the effects of DCK on various malignant biological behaviors of hepatoma cell lines were studied.The effects of DCK on the tumorigenicity of HepG2 cells in vivo were evaluated by nude mouse tumorigenesis experiment.The molecular mechanism of DCK regulating the occurrence and progress of hepatoma was preliminarily explored.Part Ⅰ Screening of candidate immune-related genes for HCC Objectives:Based on the TCGA database and ImmPort database,the bioinformatics analysis was used to screen out differentially expressed immune-related genes between HCC tissues and adjacent tissues,and then excavate immune-related genes that have prognostic value for patients with HCC.Methods:1.Screening differentially expressed immune-related genes in HCC tissues using TCGA database and ImmPort database.Gene expression data and clinical data of HCC patients were downloaded from TCGA database.Then we downloaded 1811 immune-related genes in the ImmPort database.In R software,the Wilcoxon test was used to detect differentially expressed immune-related genes in HCC tissues and adjacent tissues.The potential biological functions of differentially expressed immune-related genes were evaluated.2.Screening for differentially expressed immune-related genes related to survival.Survival analysis was performed only in patients with HCC who were followed up for less than 2,000 days.In R software,Kaplan-Meier survival analysis method and Cox method were used to search for differentially expressed immune-related genes related to survival,respectively,and P value less than 0.01 was used as the filtration criteria.After searching by the two methods,the co-existence of differentially expressed immune-related genes with prognostic value was retained.3.Screening of independent prognostic factors.Multivariable Cox analysis was performed for each gene obtained in the previous step along with clinical data(age,grade,T stage,etc.),and a P value of less than 0.01 was used as a screening condition to screen independent prognostic immune-related genes.4.Predicting analysis of transcription factors that regulate immune-related genes for independent prognosis of HCC.In order to study the regulatory mechanism of immune-related genes,we extracted 318 transcription factors from the Cistrome cancer database for further research.We used TCGA HCC data to find differentially expressed transcription factors and analyze their survival.Then we used contest function in R to get the correlation between survival related TFs and prognostic immune-related genes.The filtering criteria were correlation coefficients greater than 0.5 and P values less than 0.05.Cytoscape software was applied to build and visualize regulatory networks.Results:1.Results of differentially expressed immune-related genes in HCC tissues.The pre-processed RNA-Seq-FPKM data of 374 cases of HCC and 50 cases of adjacent tissues were downloaded from the TCGA database for further analysis.A total of 329 immune-related genes(267 up-regulated and 62 down-regulated)were differentially expressed in HCC tissues compared with adjacent tissues.The biological functions of 329 differentially expressed immune-related genes were further analyzed by GO and KEGG analysis.2.Screening of survival-related differentially expressed immune-related genes.We used the 329 differentially expressed immune-related genes from the differential analysis for survival analysis.According to the original data,a total of 337 HCC patients who were followed up for less than 2000 days were found.Univariate Cox analysis was used to detect the expression of 329 differential immune-related genes in 337 HCC tissues,and a total of 64 survival-related immune-related genes were found.The Kaplan-Meier method was used to analyze 58 survival-related immune-related genes.Using the Venn diagram,32 coexisting survival-related immune-related genes were found,all of which were high-risk genes.3.Results of screening independent prognosis of immune-related genes.Independent prognosis analysis was performed on the 32 immune-related genes obtained above,and 12 immune-related genes were obtained as independent prognostic factors.Among them,DCK was abnormally expressed in a variety of malignant tumors and participated in the initiation and progression of tumors.However,the specific mechanism of DCK in HCC has not been elucidated,which needs to be further explored.4.Construction of regulatory network of transcription factors and independent prognostic immune-related genes.Compared with adjacent tissues,a total of 117 transcription factors(108 up-regulated and 9 down-regulated)were differentially expressed in HCC tissues.Using univariate Cox analysis and Kaplan-Meier method analysis,27 survival-related transcription factors were obtained.According to the filtering criteria,a total of 25 survival related transcription factors and nine prognostic immune-related genes were identified to establish a network.Among them,HCFC1 regulated most immune-related genes.We predicted that the most important transcription factor upstream of DCK was HCFC1.Conclusions:1.Through the analysis of TCGA and ImmPort database,we found that 12 differentially expressed immune-related genes were closely related to the prognosis of HCC,and could independently predict the prognosis.Among them,the biological role of DCK in HCC is not clear,and DCK may be the key immune-related gene of HCC.2.It was predicted that 25 transcription factors could regulate one or more immune-related genes,among which HCFC1 played an important regulatory role in HCC,and it was preliminarily confirmed that HCFC1 was the most important upstream regulatory transcription factor of DCK.Part Ⅱ The expression of DCK in hepatocellular carcinoma and its correlation with pathological parameters in HBV-related hepatocellular carcinomaObjectives:The expression of DCK in HCC was determined by several open databases.We explored the relationship between DCK and immune microenvironment,and predicted the related pathways of DCK regulation in HCC.We detected the expression of DCK in HBV-related hepatocellular carcinoma.Then we studied the relationship between DCK expression and the clinicopathological characteristics of HBV-related hepatocellular carcinoma.The expression levels of DCK mRNA and protein in different human hepatoma cell lines(HepG2,SKHEP1)and normal hepatocytes HL-7702 were detected.Methods:1.We queried the expression of DCK in HCC tissues and corresponding normal tissues through the oncomine database,GEPIA website and ICGC database.2.We used TIMER website to investigate the relationship between DCK and tumor immune microenvironment in TCGA HCC patients;Then we used GSEA method to predict the related pathways of DCK regulation in HCC.3. QRT-PCR was used to detect the expression of DCK in 53 cases of HBV-related HCC tissues and adjacent tissues,and the relevance between DCK and clinicopathological parameters was analyzed.4.QRT-PCR and Western blot were used to detect the expression of DCK in different human hepatoma cell lines(HepG2,SKHEP1)and normal liver cells HL-7702.Results:1.The results of ICGC,oncomine and GEPIA suggested that DCK expression was higher in HCC tissues.2.DCK expression was positively correlated with CD4+ T cells,neutrophils,dendritic cells,B cells,macrophages and CD8+ T cells in the tumor immune microenvironment.GSEA shows that DCK can positively regulate a variety of pathways to affect the occurrence and development of HCC, including WNT signaling pathway and ERBB signaling pathway.3.QRT-PCR detection showed that the expression level of DCK in HBV-related hepatocellular carcinoma tissues were significantly higher than that in adjacent tissues.The expression of DCK was not correlated with age and gender,but correlated with tumor size,Edmondson grade,microvascular invasion.4.The expression levels of DCK mRNA and protein in HepG2 and SKHEP1 were higher than those in normal hepatocytes.Conclusions:DCK was highly expressed in HCC tissues.The expression level of DCK was positively correlated with immune ceils in immune microenvironment,indicating that DCK could reflect the status of immune microenvironment.DCK was highly expressed in HBV-related hepatocellular carcinoma tissues,and associated with a variety of clinicopathological parameters.DCK was highly expressed in HepG2 and SKHEP1,which can be used in subsequent experiments.Part Ⅲ The malignant biological characteristics of hepatoma cells promoted by DCKObjectives:By interfering with the expression of DCK in HepG2 and SKHEP1 cells,we detected the effect of DCK on the proliferation,apoptosis,invasion and other malignant biological behaviors of hepatoma cells,and fiirther verified it by nude mice tumor formation experiment.We preliminarily discussed the molecular mechanism of DCK promoting the occurrence and progress of HCC.Methods:QRT-PCR and Western blot were used to screen the most efficient sequence.Transwell invasion test,wound healing test,CCK-8 proliferation test and flow cytometry were used to examine the migration,invasion,proliferation and apoptosis of HCC cells after low expression of DCK gene.Nude mice were subcutaneously injected with HepG2 of siRNA DCK group and negative control group.After 4 weeks,the nude mice were killed,and the tumor was stripped and weighed.Western blot and cellular immunofluorescence were used to detect the effect of DCK on Wnt-I β-catenin and c-myc.Results:One siRNA was successfully screened for subsequent experiments.CCK-8 assay showed that the proliferation of HepG2 and SKHEP1 cells was significantly decreased.Cell scratch test and Transwell invasion assay showed that HepG2 and SKHEP1 cells with silenced expression of DCK exhibited significantly attenuated migration and invasion.Flow cytometry analysis showed that DCK down-regulation could promote the apoptosis of hepatoma cells.The results of animal experiments showed that the growth rate of tumor in DCK interference group was slow,and the mass and volume of tumor decreased significantly.After inhibiting the expression of DCK,the protein expression of WNT signaling pathway decreased.This suggested that DCK abnormally activated WNT signaling pathway,which enables increased the expression of key signaling molecules Wnt-1,β-catenin and c,myc,and promoted the malignant process of hepatocellular carcinoma.Conclusions:In vitro,silencing DCK gene inhibited the proliferation,migration and invasion of hepatoma cells HepG2 and SKHEP1,and promoted the apoptosis of hepatoma cells.Animal experiments showed that,silencing DCK significantly inhibited the tumorigenicity in nude mice.DCK may afifect the malignant biological behavior of HCC by activating WNT signaling pathway.DCK is expected to be a new target for Ihe treatment of HCC.更多还原