【作者】 王媛；

【导师】 赵彦艳；

【作者基本信息】 中国医科大学 ， 遗传学， 2022， 博士

【摘要】 目的：食道癌(esophageal cancer,EC)是世界范围内常见的威胁人类健康的恶性肿瘤之一,在最常见的癌症中位列第八,在癌症相关死亡中位列第六。EC主要有两个亚型:食道鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)和食道腺癌(esophageal adenocarcinoma,EAC),其中ESCC是主要的组织学类型,约占90%。ESCC起病隐匿,早期诊断率低,具有局部浸润性生长、早期发生淋巴结转移及血管侵袭等特点,尽管可以手术治疗或放化疗,ESCC的预后不佳,整体5年生存率仅有15-25%,因此,我们需要更清楚地了解ESCC发病机制,找到无创、高效、灵敏的早期筛查标记物,以争取提高早期诊断率,选择合适的有针对性的治疗手段以改善预后。已有研究证实异常的遗传学及表观遗传学变化参与了ESCC的发生发展。DNA甲基化,组蛋白修饰和功能性非编码RNA是常见的表观遗传修饰,可以调控基因表达,其中研究最多最广泛的是DNA甲基化,即在DNA甲基转移酶的作用下,在基因组启动子区域CpG(cytosin-phosphate-guanosine)二核苷酸的胞嘧啶第五碳位共价键结合一个甲基基团。在脊椎动物中,约50%基因的启动子上有CpG岛,该区域常发生异常高甲基化,引起基因表达及生理功能的异常。深入的肿瘤学和表观遗传学研究发现,抑癌基因可因启动子甲基化表达沉默而促进肿瘤的发生发展。基因芯片技术是90年代中期以来飞速发展起来的分子生物学高新技术。DNA甲基化检测手段历经了从最初的甲基化特异性PCR针对单个候选基因的检测到利用甲基化芯片对疾病的全基因组DNA甲基化水平进行宏观检测,目前的研究中应用比较广泛的是Illuminate平台的Illumina Human Methylation EPIC Bead Chip(850k)芯片,该芯片是用于全表观基因组关联研究的重要手段,覆盖超过85万个CpG位点,在单核苷酸水平上对整个基因组的甲基化位点进行定量检测。Septin属于丝状蛋白家族,该家族基因最早被发现于酿酒酵母中,在人类中Septin家族包括13个成员。SEPT9作为该家族中的一员,位于人类染色体17q25.3,普遍存在于真核生物中,参与细胞骨架重构、细胞质分裂、微管调节、囊泡运输及胞外分泌作用。在不同肿瘤中SEPT9表达水平和生理功能各不相同,并且有多个研究发现SEPT9在恶性肿瘤中存在甲基化异常,而其在ESCC中的表达和作用报道甚少。外周血cfDNA(cell free DNA)检测用于肿瘤的无创诊断技术,目前主要应用于癌基因及抑癌基因突变的检测,临床常用的检测基因包括EGFR,APC,HRAS,MET,TP53,KRAS,BARF等。已有研究证实外周血cfDNA异常的甲基化可以作为恶性肿瘤早期、无创筛查的新指标,多篇文献报道了ESCC组织中存在多种基因的过甲基化表达,然而ESCC外周血cfDNA甲基化情况报道很少。本研究中我们拟利用Illumina平台850k芯片对ESCC组织进行差异甲基化位点及区域进行筛选,根据筛选结果验证目标基因在ESCC中的甲基化及表达情况,并进一步探究其参与ESCC发生发展的机制。研究方法：1、实验材料:(1)ESCC及癌旁新鲜冰冻组织。(2)ESCC患者外周血及对照组病例外周血。(3)人类食道鳞状细胞癌KYSE-150、TE-1细胞系。(4)Real-time PCR相关试剂及仪器。(5)Western印迹相关试剂及仪器。(6)细胞培养所需试剂及仪器。(7)SEPT9表达载体:SEPT9过表达质粒。(8)组织、外周血DNA提取试剂盒。(9)免疫组织化学相关试剂、仪器。2、实验方法:(1)采用Illumina Human Methylation EPIC Bead Chip(850k)芯片对4对ESCC及癌旁组织的差异甲基化位点及区域进行筛查,结合UALCAN数据库及文献信息对SEPT9在ESCC中的表达及甲基化水平进行验证。(2)Real-time PCR方法检测ESCC组织及癌旁正常食道组织中、ESCC患者及对照组患者外周血中SEPT9甲基化水平。(3)Western Blot检测ESCC及癌旁组织中SEPT9蛋白质的表达水平。(4)免疫组织化学方法检测ESCC及癌旁组织中SEPT9蛋白质的表达情况。(5)构建SEPT9过表达质粒,转染ESCC细胞系KYSE-150、TE-1。(6)利用划痕愈合实验、Transwell实验、CCK-8、细胞凋亡实验鉴定SEPT9在细胞迁移、侵袭、增殖和凋亡中的作用。(7)Western Blot检测转染细胞GSK-3β磷酸化蛋白水平变化,鉴定GSK-3β活性是否改变。(8)Western Blot检测转染细胞核内β-catenin蛋白表达,验证过表达SEPT9能否导致β-catenin核转位异常;加入GSK-3β活性抑制剂(TWS119),鉴定β-catenin蛋白表达是否回调。结果：1、采用Illumina Human Methylation EPIC Bead Chip(850k)芯片对4对ESCC组织及配对癌旁组织进行差异甲基化位点及区域筛查,共筛查出差异甲基化位点189489个,其中高甲基化位点53307个,低甲基化位点136182个,差异甲基化区域261个。选取排名前30的区域,共有34个基因位于其中,整合公共数据库UALCAN中的ESCC甲基化芯片数据,筛选出6个基因:ZNF132、SEPT9、ZIC1、ZIC4、USP4以及ZNF154。查找以上6个基因甲基化差异最明显的探针位点,并将其在所有筛选出的位点中按照甲基化程度的降序进行排序,发现SEPT9的代表位点cg20309069在所有筛选出的189489个位点中位列18,位列上述6个基因中的首位,拟在下一步研究中验证SEPT9在ESCC中的甲基化及表达情况。2、在31对ESCC组织及癌旁组织中进行SEPT9甲基化检测,发现SEPT9在ESCC组织中甲基化水平(1.20±1.92)较癌旁食道组织(3.64±1.92)明显升高,绘制ROC曲线,曲线下面积0.8293。3、在外周血水平,我们发现81%(51/63)的ESCC患者和6%(4/71)的对照组患者存在异常升高的SEPT9甲基化,ESCC组患者的甲基化水平(10.42±4.36)较对照组(16.72±1.68)病例明显升高,绘制ROC曲线,曲线下面积0.8390。4、对63例ESCC患者的临床数据进行分组分析,发现外周血游离DNA中SEPT9异常升高的甲基化与ESCC的淋巴结转移、TNM分期相关,且SEPT9甲基化发生在肿瘤形成早期。5、Western Blot实验证实了SEPT9蛋白在ESCC组织中的表达(0.1804±0.0630)较癌旁正常食道组织(0.8431±0.1596)降低;免疫组化实验证实在200倍视野下,ESCC中(3.7587±1.3241%)SEPT9阳性面积百分比较癌旁组织(54.4580±2.6059)明显降低。6、构建SEPT9过表达载体,分别转染ESCC细胞系KYSE150及TE-1,通过划痕愈合实验、Transwell实验、CCK-8实验及凋亡实验证实,过表达SEPT9抑制ESCC细胞的迁移、侵袭和增殖能力,促进其凋亡。7、转染ESCC细胞系SEPT9过表达质粒及空载体Vector,结果证实,在KYSE-150及TE-1细胞系中,过表达组的GSK-3β在Ser9位点表达水平较Vector组降低,说明SEPT9过表达增强了GSK-3β的激酶活性。8、ESCC细胞系转染SEPT9过表达质粒后,细胞核内β-catenin蛋白质水平的表达明显降低,在加入TWS119后,细胞核β-catenin蛋白表达水平出现回复。结论：1、ESCC患者外周血中存在着与组织中相一致的SEPT9甲基化水平升高,并且是肿瘤发生的早期事件,外周血游离DNA中甲基化水平的高低与ESCC是否合并淋巴结转移及TNM分期相关,为ESCC的早期、无创、高效筛查提供了一种新的可能。2、我们的研究数据表明SEPT9蛋白质在ESCC组织中低表达,这可能是由于SEPT9在ESCC组织中过度甲基化造成的。3、过表达SEPT9可抑制ESCC细胞增殖、转移和侵袭能力,但促进其凋亡,在ESCC中它可能是通过调节GSK-3β的活性起到抑制Wnt/β-catenin信号通路从而发挥抑癌基因的作用。更多还原

【Abstract】 Objective:Esophageal cancer is one of the most common malignancies worldwide.It ranks sixth in leading cause of cancer related mortality.Esophageal cancer is subdivided into two main histological types:esophageal squamous cell carcinoma（ESCC）and esophageal adenocarcinoma（EAC）,and about 90%of the patients are ESCC.ESCC is characterized by aggressive local growth,early lymphatic metastasis and vascular invasion.Despite the improvement of surgery and chemoradiotherapy,the prognosis of ESCC is poor due to asymptomatic progression in early stage and diagnosis in advanced stage.The overall 5-year survival is merely about 15-25%.It is high time to develop sensitive,specific and none-invasive biomarkers for early diagnosis to improve the overall prognosis for ESCC.It is demonstrated that aberrant genetic and epigenetic changes lead to the pathogenesis and progression of human ESCC.There are three kinds of familiar epigenetic modifications:histone modification,DNA methylation and non-coding RNA.DNA methylation is a common epigenetic modification and plays a crucial role in tumor genesis by regulating gene expression without changing the DNA sequence.Aberrant DNA methylation usually occurs on cytosin-phosphate-guanosine islands（CpG islands）of gene promoter region which involved in the occurrence and development of ESCC.With the in-depth research in oncology and epigenetics,we find promotor methylation of tumor suppressor gene silence the expression of the protein which promotes the development of malignant tumors.Gene microarray technology is a high-tech in molecular biology that has developed rapidly since the mid-1990s.DNA methylation detection have experienced from the detection of a single candidate gene by methylation specific PCR to the detection of the whole genome DNA methylation level by methylation chip.The Illumina Human Methylation EPIC Bead Chip（850k）is widely used in researches nowadays which is an important means for whole epigenome association research.The chip covers more than 850,000 CpG sites and quantitatively detects the methylation sites of the whole genome at the level of single nucleotide.Septin belongs to the filamentous protein family.The gene was first found in Saccharomyces cerevisiae.The septin gene family includes 13 members in humans.Septin 9（SEPT9）gene is located at chromosome 17q25.3 and ubiquitous in eukaryotes.It encodes SEPT9 protein which plays important physiological role in cytoskeletal remodeling,actin dynamics,microtubule regulation,vesicle trafficking and exocytosis.It is abnormally expressed in various kinds of cancer with different biological effects.Peripheral cell free DNA detection is used for non-invasive diagnosis of tumors.At present,it is mainly used for the detection of mutations in oncogene and tumor suppressor genes.EGFR,APC,MET,TP53,KRAS and BARF are common detected genes in clinics.Previous studies have confirmed that the abnormal methylation of cell free DNA in peripheral blood can be used as a new biomarker for early and noninvasive screening of malignant tumors.Many studies have reported the hypermethylation expression of many genes in ESCC tissues.However,there are few reports on the methylation of cell free DNA in plasm of ESCC.In our study,we intend to screen the differentially methylated sites and regions of ESCC tissues by using Illumina Human Methylation EPIC Bead Chip （850k）.Then we try to verify the methylation and expression of target gene in ESCC according to the screening results.Furthermore,we try to uncover the mechanism of how the target gene participating in the development and occurrence of ESCC.Materials and Methods:1.Materials（1）Fresh frozen esophageal squamous cell carcinoma tissue and paracancerous tissue.（2）Blood specimens from esophageal squamous cell carcinoma patients and control cases.（3）Esophageal squamous cell carcinoma cell line KYSE-150 and TE-1.（4）Real-time PCR experiment reagents and instruments.（5）Western blot experiment reagents and instruments.（6）Cell culture related reagents and instruments.（7）SEPT9 over-expression vector.（8）Immunohistochemical related reagents.2.Methods:（1）Illumina human methylation epic beadchip（850k）chip was used to scan the differential methylation sites and regions of 4 pairs of ESCC and adjacent tissues.Combined the 850k database with UALCAN database of tumor methylation,the expression and methylation level of SEPT9 in ESCC were verified.（2）Real-time PCR to conform the methylation level in ESCC patients and control cases.Analyze the clinical information of ESCC cases to explore the relationship between clinical characters and methylation.（3）Western blot and immunohistochemistry experiments to confirm the expression of SEPT9 in ESCC tissue and para-cancer tissue.Real-time PCR to conform the methylation level in ESCC tissue and para-cancer tissue.（4）Build SEPT9 over-expression plasmid and transfect ESCC cell lines KYSE-150and TE-1.（5）Wound healing,transwell,CCK-8 and apoptosis experiments to confirm the impact of SEPT9 on ESCC cell lines.（6）Western blot experiment to compare the expression of phosphorylated GSK-3βin SEPT9 over-expression group and vector group.（7）Western blot experiment to compare the expression ofβ-catenin in SEPT9 over-expression group and vector group in nuclear protein.Then confirm whether GSK-3βinhibitor TWS119 could revert the expression ofβ-catenin.Results:1.4 pairs of ESCC tissues and para-cancer tissues were scanned with Illumina Human Methylation EPIC Bead Chip（850k）.We found 189489 differential methylation sites.There were 53307 hypermethylation sites and 136182 hypomethylation sites.Furthermore,we found 261 differential methylation regions.We chose top 30 differential methylation regions and found 34 genes located in these regions.The methylation chip information from UALCAN database were integrated with our screening database.We found 6 genes including ZNF132,SEPT9,ZIC1,ZIC4,USP4and ZNF154 possessed hypermethylation in both databases.Found the probe site which had the highest hypermethylation level among all probe sites of the gene to be the representative site.Sort the results in descending order among all 189489 differential methylation sites.The representative site cg20309069 of SEPT9 ranked18^th among all sites which was the first among the six genes.Therefore,we choose to confirm the expression and methylation of SEPT9 in ESCC.2.The methylation of SEPT9 was increased in ESCC tissue（1.20±1.92）than para-cancer tissue（3.64±1.92）.3.81%（51/63）of esophageal squamous cell carcinoma patients and 6%（4/71）of control cases had SEPT9 hypermethylation in plasm cell free DNA.Plasm cell free DNA from esophageal squamous cell carcinoma patients（10.42±4.36）had an obviously higher methylation level than control cases（16.72±1.68）.4.The methylation of SEPT9 was correlated with TNM stage and lymphatic metastasis.Hypermethylation occurred early in ESCC.5.The expression of SEPT9 was decreased in ESCC tissue than para-cancer tissue.6.Over-expression of SEPT9 inhibited migration,invasion,proliferation abilities of ESCC cell while promotes apoptosis abilities.7.Over-expression of SEPT9 inhibited the expression of phosphorylated GSK-3βwhich promoted the kinase activity of GSK-3β.8.Over-expression of SEPT9 inhibited the expression ofβ-catenin in nuclear while the expression was reverted when TWS119 was added.Conclusion:Our study suggested the expression of SEPT9 was decreased in ESCC which may due to the hypermethylation of the gene.Plasm from esophageal squamous cell carcinoma patients had an distinctly higher methylation status of SEPT9 than control cases which is parallel with ESCC tissue.The methylation of SEPT9 was correlated with TNM stage and lymphatic metastasis.Hypermethylation occurred early in ESCC.Plasm SEPT9 hypermethylation could be a potential biomarker for none-invasive and early screening of ESCC.SEPT9 may acts as a tumor suppressor gene in ESCC by adjusting the activity of GSK-3βto inhibit Wnt/β-catenin signaling pathway.更多还原