【作者】 王凯；

【导师】 杜贾军；

【作者基本信息】 山东大学 ， 外科学（胸心外科）（专业学位）， 2024， 博士

【摘要】 肺癌是我国2022年发病率及死亡率最高的恶性肿瘤,发病占所有新发恶性肿瘤约22.0%,死亡占所有恶性肿瘤约28.5%,且在男性及女性中均居首位。肺癌按照病理类型分为非小细胞肺癌(约85%)及小细胞肺癌。由于病理学形态、分子特征、发病机制、治疗手段及对治疗反应等方面存在较大差异,非小细胞肺癌主要分为肺腺癌及肺鳞癌等病理类型。近年来随着CT筛查的普及,肺腺癌的早期检出比例较既往显著升高。尽管肺癌的治疗手段包括手术治疗、放化疗、免疫治疗、靶向治疗等不断推陈出新,但总体五年生存率仍不足20%。探索肺腺癌的发生发展机制有利于发现新的治疗靶点,对于改善和提高肺腺癌患者的长期生存及生活质量至关重要。ZBTB16(Zinc Finger And BTB Domain-Containing Protein 16),兼有 E3 泛素连接酶和转录因子的功能。ZBTB16为真核细胞中高度保守的蛋白,由673个氨基酸构成。ZBTB16最早被发现是因其与RARα形成ZBTB16-RARα融合蛋白导致早幼粒白血病的发生及耐药,因而也被名为PLZF(Promyelocytic Leukemia Zinc Finger,早幼粒白血病锌指蛋白)。ZBTB16作为ZBTB16-Cullin3-Roc1 E3泛素连接酶的成员,可特异性识别底物蛋白Atg14L并通过泛素蛋白酶体途径促进其降解从而调控细胞自噬。ZBTB16在更多的研究中作为转录因子发挥作用,其N端为BTB/POZ功能结构域,可与其他蛋白相互作用并组建成转录抑制复合物。而C端为Kruppel样C2H2锌指结构域,可以发挥结合靶基因的作用。而锌指结构域的乙酰化及BTB/POZ与锌指结构域中间位点的SUMO化为其发挥转录因子功能所必需。ZBTB16作为转录因子参与和调控干细胞维持、肢体发育、精子发生、免疫细胞分化和发育及肿瘤的发生发展等。在绝大部分恶性肿瘤的研究中,ZBTB16发挥广泛的抑癌作用,包括抑制细胞增殖、促进细胞凋亡、抑制血管生成及抑制肿瘤转移等。近年来研究表明,ZBTB16在非小细胞肺癌、胰腺癌、乳腺癌、前列腺癌、肝细胞肝癌、胆囊癌等诸多实体肿瘤中显著低表达,并发挥抑制细胞增殖的作用。且在非小细胞肺癌、胰腺癌等研究中发现肿瘤组织内ZBTB16启动子区域存在甲基化修饰,并导致其在肿瘤中表达下调。目前ZBTB16作为转录因子已报道的靶基因包括 ACTA2、BID、CCNA2、IFIT2、ID2、Hox、KIT、MYC 等,发挥转录抑制或转录激活功能因靶点及特定生物学行为等而异。然而在肺腺癌中ZBTB16发挥作用的身份、调控机制及靶点等尚不明确。本研究通过生物信息学技术分析公共数据库中ZBTB16在肺腺癌中表达和预后价值,ZBTB16启动子区域的甲基化水平及对其表达的影响。并通过临床样本进行甲基化、表达差异及预后影响的验证。通过SRB染色细胞增殖、EdU染色、PI细胞周期实验、Annexin V-FITC/PI细胞凋亡实验、Transwell侵袭迁移实验等体外实验探索ZBTB16对肺腺癌细胞增殖、细胞周期、细胞凋亡、侵袭迁移等肿瘤生物学行为的调控作用。通过非标记定量质谱、染色质免疫共沉淀测序、RNA测序等技术探索ZBTB16发挥上述调控作用的身份和相应靶点,继而通过裸鼠皮下成瘤进行体内实验验证。我们发现,ZBTB16在肺腺癌中显著低表达,且肺腺癌中低表达与不良预后相关。肺腺癌组织ZBTB16启动子存在甲基化修饰,将细胞系进行去甲基化处理后ZBTB16表达回复,提示DNA甲基化为ZBTB16在肺腺癌中表达下调的重要原因之一。ZBTB16主要抑制DNA复制及细胞增殖,并可引起细胞周期阻滞。而发挥相应功能主要通过转录因子的身份,通过ChIP-seq、RNA-seq、TCGA-LUAD等结果综合分析筛选出其潜在的关键转录靶点WDHD1。通过表达相关性、双荧光素酶报告基因技术验证物理层面结合、挽救实验进行功能学验证等多维度进行了相应的验证,我们发现ZBTB16通过结合WDHD1启动子区域抑制其转录进而抑制肺腺癌细胞DNA复制及阻滞细胞周期,并通过裸鼠皮下成瘤实验验证了上述体外实验结果。本研究旨在阐明ZBTB16在肺腺癌中的表达预后意义及发挥抑癌作用的具体机制,为肺腺癌的治疗提供新的研究方向。第一部分ZBTB16在肺腺癌中的表达差异及对预后的影响研究目的1.研究ZBTB16在肺腺癌及正常肺组织中的表达差异。2.探讨肺腺癌组织中ZBTB16表达对患者长期预后的影响。3.明确肺腺癌组织中ZBTB16启动子区域甲基化修饰情况及对表达的影响。研究方法1.利用生物信息学技术研究和筛选肺腺癌与正常肺组织中差异表达的E3泛素连接酶,其中差异最显著的为ZBTB16。利用公共数据库TCGA、GEO及KMplotter工具研究ZBTB16对肺腺癌患者总生存期的影响。分析TCGA及GEO中肺腺癌甲基化芯片明确ZBTB16启动子区域甲基化修饰水平。并通过泛癌分析明确多种实体肿瘤中ZBTB16表达及启动子甲基化修饰情况。2.收集肺腺癌手术切除的新鲜冰冻组织,利用RT-qPCR技术检测ZBTB16的mRNA表达水平,分析其在肿瘤与癌旁组织中表达差异及对患者总生存期的影响。3.收集肺腺癌手术切除组织的病理切片并结合组织芯片分析,利用免疫组织化学染色示ZBTB16蛋白表达水平,分析其对肺腺癌患者总生存期的影响。4.收集肺腺癌手术切除的新鲜组织,进行甲基化特异性PCR(MSP)及凝胶电泳实验检测ZBTB16启动子区域的甲基化修饰水平。5.去甲基化处理肺腺癌细胞系及正常肺上皮细胞系,利用MSP、PCR及凝胶电泳检测ZBTB16启动子甲基化水平变化及对表达的影响。结果1.通过对TCGA-GTEx及10个独立包含肺腺癌及正常肺组织的GEO数据集分别进行差异分析,结果显示与正常肺组织相比,在所有11个独立的肺腺癌数据集中肿瘤组织中ZBTB16 mRNA水平均显著降低(p<0.001)。而KMplotter、TCGA及GEO联合预后分析结果均显示低表达ZBTB16的肺腺癌患者总生存期较短(p<0.05)。2.RT-qPCR结果验证上述生信分析ZBTB16在肺腺癌组织中表达下调且与预后密切相关。3.免疫组织化学染色结果显示低表达ZBTB16与不良预后相关(p=0.031)。4.公共数据库甲基化芯片及新鲜组织MSP结果显示肿瘤组织中ZBTB16启动子甲基化水平较高。而肺腺癌细胞系去甲基化处理后ZBTB16启动子甲基化水平下降伴随mRNA水平升高,提示启动子区域DNA甲基化参与ZBTB16在肺腺癌中的表达下调。结论1.ZBTB16在肺腺癌组织中表达下调,且ZBTB16表达水平与预后密切相关。2.ZBTB16启动子区域存在甲基化修饰,甲基化修饰参与其在肺腺癌组织中的表达下调。第二部分ZBTB16对肺腺癌DNA复制及细胞周期的调控作用及机制研究研究目的1.探索ZBTB16对肺腺癌细胞的调控作用。2.明确ZBTB16在肺腺癌中发挥关键作用的身份。3.筛选和验证ZBTB16的作用靶点及结合位点。4.体内实验进行上述体外实验结果的验证。研究方法1.通过RNA-seq技术筛选过表达ZBTB16组与对照组的差异基因。2.通过生物信息学技术分析TCGA-LUAD高低表达ZBTB16组差异基因及RNA-seq结果差异基因进行通路富集分析探索潜在的研究方向。3.通过SRB染色细胞、EdU染色、PI周期染色、Annexin V-FITC/PI染色、免疫荧光染色、Transwell侵袭迁移实验等体外实验探讨ZBTB16对细胞增殖、DNA复制、细胞凋亡、细胞周期、侵袭迁移等的调控作用。4.通过免疫共沉淀联合非标记定量质谱技术、染色质免疫共沉淀测序(ChIP-seq技术)技术分别探讨ZBTB16的互作蛋白以及ZBTB16结合的DNA序列。5.通过双荧光素酶报告基因技术验证ZBTB16与WDHD1启动子区域的直接结合,并发挥转录抑制作用。6.通过挽救实验验证WDHD1为ZBTB16抑制DNA复制及阻滞细胞周期的功能学靶点。7.通过裸鼠皮下成瘤实验验证ZBTB16对肺腺癌细胞增殖的抑制作用及对细胞周期相关蛋白表达的影响。结果1.RNA-seq及TCGA-LUAD中富集分析结果显示ZBTB16主要调控DNA复制及细胞周期。另外,GO分析显示其还与有丝分裂相关。2.体外实验结果显示ZBTB16可显著抑制肺腺癌细胞增殖及DNA复制,引起细胞周期的S期比例升高,抑制侵袭迁移能力,并促进细胞凋亡、DNA损伤及有丝分裂崩溃。3.非标记定量质谱结果显示与ZBTB16互作的蛋白主要参与RNA剪切及蛋白翻译。ChIP-seq结果显示ZBTB16结合的DNA与RNA-seq差异基因、TCGA-LUAD差异基因和细胞周期基因集取交集鉴定潜在的靶基因为WDHD1。4.双荧光素酶报告基因实验验证ZBTB16与WDHD1启动子区域相结合,以及ZBTB16对WDHD1的转录抑制作用。5.挽救实验验证WDHD1可部分弥补ZBTB16对细胞增殖和DNA复制的抑制作用及对细胞周期的阻滞作用。6.针对既往研究中ZBTB16调控S期的潜在靶点Cyclin A2(CCNA2)进行表达抑制和挽救实验的验证。7.体内实验验证ZBTB16对肿瘤增殖的抑制作用。结论1.ZBTB16可显著抑制肺腺癌细胞增殖及DNA复制,并引起细胞周期阻滞。2.ZBTB16作为转录因子靶向抑制WDHD1转录发挥抑制DNA复制及阻滞细胞周期的作用。3.研究ZBTB16在肺腺癌中表达下调机制及对DNA复制、细胞周期的调控机制,有望成为肺腺癌诊断和治疗的新策略新方向。综述ZBTB16表达及功能调控机制的研究进展本部分重点总结归纳了 ZBTB16表达的上游调控机制及蛋白翻译后修饰,主要包括ZBTB16基因突变、染色体融合、可变剪切、截短蛋白、DNA甲基化、组蛋白甲基化、组蛋白乙酰化、非编码RNA调控、蛋白磷酸化、蛋白乙酰化、蛋白SUMO化及蛋白泛素化。还简要介绍了 ZBTB16存在互作的蛋白及作为转录因子直接结合和调控的靶基因。更多还原

【Abstract】 Lung cancer is the malignant tumor with the highest incidence and mortality in China in 2022,accounting for about 22.0%of all new cancer cases and 28.5%of all cancer deaths,and ranking first in both men and women.Lung cancer is divided into non-small cell lung cancer(about 85%)and small cell lung cancer(SCLC)according to pathological types.Non-small cell lung cancer is mainly divided into lung adenocarcinoma and lung squamous cell carcinoma due to the great differences in pathological morphology,molecular characteristics,pathogenesis,treatment options and response to treatment.In recent years,with the popularization of CT screening,the early detection rate of lung adenocarcinoma has increased significantly.Although the treatment methods of lung cancer include surgery,radiotherapy,chemotherapy,immunotherapy,targeted therapy and so on,the overall five-year survival rate is still less than 20%.Exploring the pathogenesis of lung adenocarcinoma is conducive to the discovery of new therapeutic targets,which is very important for improving the long-term survival and quality of life of patients with lung adenocarcinoma.ZBTB16(Zinc Finger And BTB Domain-Containing Protein 16)acts as both an E3 ubiquitin ligase and a transcription factor.ZBTB16 is a 673 amino acid protein that is highly conserved in eukaryotic cells.ZBTB16 was first discovered to form ZBTB16-RARα fusion protein and lead to the development and drug resistance of promyelocytic leukemia,so it was also named PLZF(Promyelocytic Leukemia Zinc Finger protein).As a member of ZBTB16-Cullin3-ROC1 E3 ubiquitin ligase,ZBTB16 can specifically recognize the substrate protein Atg14L and promote its degradation through the ubiquitin proteasome pathway to regulate cell autophagy.In most studies,ZBTB16 functions as a transcription factor with a BTB/POZ functional domain at its N terminus,which can interact with other proteins and form transcription repression complexes.The Kruppel-like C2H2 zinc fingers domain at the C terminus can play a role in binding target genes.Acetylation of the zinc finger domain and SUMOylation at the intermediate site between the zinc fingers and BTB/POZ domains are required for the transcription factor function.As a transcription factor,ZBTB16 is involved in the regulation of stem cell maintenance,limb development,spermatogenesis,immune cell differentiation and development,and tumor development.In most studies of malignant tumors,ZBTB16 exerts a wide range of tumor suppressive effects,including inhibiting cell proliferation,promoting cell apoptosis,inhibiting angiogenesis and inhibiting tumor metastasis,etc.Recent studies have shown that ZBTB16 is significantly down-regulated in many solid tumors,such as non-small cell lung cancer,pancreatic cancer,breast cancer,prostate cancer,hepatocellular carcinoma,and gallbladder cancer,and plays a role in inhibiting cell proliferation.In addition,studies on non-small cell lung cancer and pancreatic cancer have found that the promoter region of ZBTB16 is hypermethylated in tumor tissues,leading to its down-regulation in tumors.At present,the reported target genes of ZBTB16 as a transcription factor include ACTA2,BID,CCNA2,IFIT2,ID2,Hox,KIT,MYC,etc.The function of transcription repression or transcription activation depends on the target genes and specific biological behaviors.However,the regulatory mechanism and targets of ZBTB 16 in lung adenocarcinoma remain unclear.The starting point of this study is to analyze the expression and prognostic value of ZBTB 16 in lung adenocarcinoma,the methylation level of ZBTB 16 promoter region and its effect on its expression by bioinformatics technology.The methylation,expression differences and prognostic effects were verified by clinical samples.SRB staining,EdU staining,PI cell cycle assay,Annexin V-FITC/PI apoptosis assay,and Transwell invasion and migration assay were used to explore the regulatory effect of ZBTB 16 on lung adenocarcinoma cell proliferation,cell cycle,cell apoptosis,invasion and migration.Label-free quantitative mass spectrometry,chromatin immunoprecipitation sequencing,and RNA sequencing were used to explore the targets of ZBTB 16,which were verified by subcutaneous tumor formation experiment in nude mice.We found that ZBTB 16 was significantly down-regulated in lung adenocarcinoma,and low expression in lung adenocarcinoma was associated with poor prognosis.Hypermethylation of ZBTB 16 promoter was found in lung adenocarcinoma tissues,and the expression of ZBTB 16 recovered after demethylation treatment in cell lines,suggesting that DNA methylation is one of the important reasons for the down-regulation of ZBTB 16 expression in lung adenocarcinoma.ZBTB 16 mainly inhibited DNA replication and cell proliferation,and caused cell cycle arrest.The potential key transcription target WDHD1 was selected by comprehensive analysis of ChIP-seq,RNA-seq,TCGA-LUAD and other results.By expression correlation analysis,dual luciferase reporter gene technique to verify physical binding,and rescue experiment to verify the function of ZBTB 16,we found that ZBTB 16 inhibited DNA replication and arrested cell cycle in lung adenocarcinoma cells by binding WDHD1 promoter region to inhibit its transcription.The results of in vitro experiments were further verified by subcutaneous tumor formation experiments in nude mice.The aim of this study is to clarify the prognostic significance of ZBTB 16 expression in lung adenocarcinoma and the specific mechanism of its tumor suppressor effect,and to provide a new research direction for the treatment of lung adenocarcinoma.Part Ⅰ:ZBTB16 expression in lung adenocarcinoma and its prognostic significance.Objectives of the Study(1)To investigate the differential expression of ZBTB16 in lung adenocarcinoma and normal lung tissues.(2)To investigate the effect of ZBTB 16 expression in lung adenocarcinoma tissues on the long-term prognosis of patients.(3)To determine the methylation status of ZBTB 16 promoter region and its effect on expression in lung adenocarcinoma tissues.Research Methods(1)After screening the differentially expressed E3 ubiquitin ligases in lung adenocarcinoma and normal lung tissues by bioinformatics technology,the most significant difference was ZBTB 16.The public databases TCGA,GEO and KMplotter tools were used to investigate the effect of ZBTB 16 on the overall survival of patients with lung adenocarcinoma.The methylation level of ZBTB 16 promoter region was determined by methylation microarray analysis in TCGA and GEO.Pan-cancer analysis was performed to determine the expression and promoter methylation of ZBTB 16 in a variety of solid tumors.(2)The expression level of ZBTB 16 mRNA was detected by RT-qPCR in the fresh frozen tissues of lung adenocarcinoma,and the difference of ZBTB 16 expression between tumor and adjacent tissues and its effect on the overall survival of patients were analyzed.(3)The expression level of ZBTB 16 protein was detected by immunohistochemical staining in tissue microarray,and its effect on the overall survival of patients with lung adenocarcinoma was analyzed.(4)ZBTB 16 promoter methylation was detected by methylation-specific PCR(MSP)and gel electrophoresis in fresh lung adenocarcinoma tissues.(5)Lung adenocarcinoma cell lines and normal lung epithelial cell lines were treated with 5-aza-2’-deoxycytidine(ADC)for demethylation,and the methylation level of ZBTB 16 promoter and its effect on expression were detected by MSP,PCR and gel electrophoresis.Results(1)Differential analysis was analyzed separately on TCGA-GTEx and 10 independent GEO datasets containing lung adenocarcinoma and normal lung tissues.The results showed that compared with normal lung tissues,ZBTB 16 mRNA levels were significantly decreased in tumor tissues in all 11 independent lung adenocarcinoma datasets(p<0.001).The combined prognostic analysis results of KMplotter,TCGA and GEO all showed that the overall survival of lung adenocarcinoma patients with low expression of ZBTB16 was shorter(p<0.05).(2)RT-qPCR results confirmed that ZBTB16 expression was down-regulated in lung adenocarcinoma tissues and was closely related to prognosis.(3)Immunohistochemical staining showed that low expression of ZBTB16 was associated with poor prognosis(p=0.031).(4)The results of methylation chip in public database and fresh tissue MSP analysis showed the hypermethylation of ZBTB16 promoter in tumor tissues.After demethylation treatment,the methylation level of ZBTB16 promoter was decreased and the mRNA level was increased in lung adenocarcinoma cell lines,suggesting that DNA methylation in the promoter region was involved in the down-regulation of ZBTB16 expression in lung adenocarcinoma.Conclusion(1)The expression of ZBTB16 is down-regulated in lung adenocarcinoma tissues,and the expression level of ZBTB16 is closely related to the prognosis.(2)The promoter region of ZBTB16 is hypermethylated,which is involved in the down-regulation of its expression in lung adenocarcinoma.Part Ⅱ:Study of the regulatory effect and mechanism of ZBTB16 on DNA replication and cell cycle in lung adenocarcinoma.Objectives of the Study(1)To explore the regulatory effect of ZBTB16 on lung adenocarcinoma cells.(2)To clarify the critical role of ZBTB16 in lung adenocarcinoma.(3)To screen and verify the target and binding sites of ZBTB16.(4)To verify the above in vitro experimental results via in vivo experiments.Research Methods(1)The differentially expressed genes between ZBTB16 overexpression group and control group were screened by RNA-seq.(2)The differentially expressed genes in the TCGA-LUAD high and low expression ZBTB16 group and the differentially expressed genes in RNA-seq results were analyzed by bioinformatics technology to explore the potential research directions.(3)SRB staining,EdU staining,PI cycle staining,Annexin V-FITC/PI staining,immunofluorescence staining,and Transwell invasion and migration assay were used to investigate the regulatory effects of ZBTB16 on cell proliferation,DNA replication,cell apoptosis,cell cycle,invasion and migration in vitro.(4)Co-immunoprecipitation combined with label-free quantitative mass spectrometry and chromatin immunoprecipitation sequencing(ChIP-seq)were used to investigate the interaction protein and the binding DNA sequence of ZBTB 16,respectively.(5)The direct binding of ZBTB 16 to the WDHD1 promoter region and its transcriptional inhibition were verified by dual luciferase reporter gene assay.(6)WDHD1 was identified as the functional target of ZBTB 16 for DNA replication inhibition and cell cycle arrest by rescue experiments.(7)The inhibitory effect of ZBTB 16 on lung adenocarcinoma cell proliferation and the expression of cell cycle related proteins were verified by subcutaneous tumor formation experiment in nude mice.Results(1)RNA-seq and TCGA-LUAD enrichment analysis showed that ZBTB 16 mainly regulated DNA replication and cell cycle.In addition,GO analysis showed that it was also associated with mitosis.(2)ZBTB 16 significantly inhibited cell proliferation and DNA replication,increased the ratio in S phase,inhibited cell invasion and migration,and promoted cell apoptosis,DNA damage and mitotic catastrophe in lung adenocarcinoma cells in vitro.(3)The results of label-free quantitative mass spectrometry showed that the proteins interacting with ZBTB 16 were mainly involved in RNA splicing and protein translation.ChIP-seq results showed that the intersection of ZBTB 16 combined with genes and RNA-seq differential genes,TCGA-LUAD differential genes and cell cycle gen set identified WDHD1 as the potential target gene.(4)Dual luciferase reporter gene assay showed the binding of ZBTB 16 to the promoter region of WDHD1 and the transcriptional inhibition effect of ZBTB 16 on WDHD1.(5)WDHD1 partially rescued the inhibitory effect of ZBTB 16 on cell proliferation,DNA replication and cell cycle arrest.(6)CCNA2,a potential target of ZBTB 16 in S-phase regulation,was also verified by expression inhibition and rescue experiments.(7)The inhibitory effect of ZBTB 16 on tumor proliferation was verified in vivo.Conclusion(1)ZBTB 16 can significantly inhibit the proliferation and DNA replication of lung adenocarcinoma cells,and cause cell cycle arrest.(2)As a transcription factor,ZBTB16 targeted WDHD1 transcription to inhibit DNA replication and arrest cell cycle.(3)Studying the down-regulation mechanism of ZBTB16 expression and its regulatory mechanism on DNA replication and cell cycle in lung adenocarcinoma is expected to become a new strategy and direction for the diagnosis and treatment of lung adenocarcinoma.Reviews:The research progress of ZBTB16 expression and functional regulation mechanism.This part mainly summarizes the upstream regulatory mechanism of ZBTB16 expression and protein post-translational modification.It mainly includes ZBTB16 gene mutation,chromosome fusion,alternative splicing,truncated protein,DNA methylation,histone methylation,histone acetylation,non-coding RNA regulation,protein phosphorylation,protein acetylation,protein SUMOylation and protein ubiquitination.We also briefly introduce the interacting proteins of ZBTB 16 and its direct binding and regulation target genes as a transcription factor.更多还原