FADS2基因在食管鳞状细胞癌中的表达及其生物学功能的初步探究

发布时间：2018-03-17 06:33

本文选题：食管鳞癌　切入点：FADS2　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的食管癌是人类常见的消化道恶性肿瘤之一,死亡率在所有肿瘤中排名第四[1],病理类型分为鳞癌和腺癌,在我国食管鳞状细胞癌(Esophageal squamous cell carcinoma,ESCC)是最主要的病理类型,其发病率具有明显的地区差异和家族聚集现象,其高发区位于东亚以及中国东部地区,特别是河南的林州、安阳、辉县和大别山区等地。当前的肿瘤分子生物学研究资料表明,环境、遗传和精神心理等因素通过协同或序贯的方式引起DNA损伤,使抑癌基因失活或者使原癌基因激活,加上凋亡调节基因或者DNA修复基因的异常改变,促使正常的食管粘膜经过一个多因素、多基因、多阶段的恶性演化过程,由此发展为食管癌[2],据此,从DNA修复基因、原癌基因、抑癌基因、代谢酶基因的遗传多态性入手研究易感基因及肿瘤标志物可以为食管癌的早期诊断、治疗及预后提供更多的理论依据[3]。有研究发现在乳腺癌组织中脂肪酸去饱和酶基因2(Fatty Acid Desaturase 2,FADS2)表达水平增高,促进亚油酸(linoleic acid,LA)转化成二十碳四烯酸(arachidonic acid,AA),同时其代谢产物前列腺素E2(Prostagland E2,PGE2)增多,在乳腺癌的发生发展中起到一定的促进的作用[4],在食管鳞癌中该基因表达水平显著上调,其机制尚不清楚。本文通过相关实验,检测FADS2基因在ESCC及癌旁正常黏膜组织中mRNA及蛋白的表达情况,分析其与患者临床病理特征和预后的关系;通过慢病毒转染系统构建稳定表达FADS2的细胞克隆,进行体外细胞增殖实验、软琼脂克隆形成实验、平板克隆形成实验、细胞迁移和侵袭实验初步探讨其生物学功能。研究方法1.临床标本的收集从河南省林州市人民医院收集手术切除的ESCC新鲜组织标本,每例标本包含食管鳞癌组织及癌旁组织,共70对,储存于液氮中,用于提取组织RNA,检测FADS2 mRNA的表达。另外,本课题组调取了河南省林州市人民医院299例ESCC根治切除术的石蜡包埋组织样本,整理临床病理和随访资料,制作ESCC的组织芯片。2.方法通过QPCR方法检测FADS2 mRNA在70例ESCC及癌旁相对正常粘膜组织标本中的表达情况;通过免疫组织化学方法检测FADS2蛋白在299对ESCC组织芯片中的表达水平,并分析FADS2的表达与ESCC患者临床病理特征和预后的相关性。通过慢病毒包装系统将FADS2表达质粒分别转染到食管癌细胞株KYSE30和KYSE510中,筛选出稳定表达FADS2的细胞,并用QPCR、Western blot鉴定。通过体外的细胞增殖实验、平板克隆形成实验、软琼脂克隆形成实验、迁移实验和侵袭实验研究FADS2对细胞生长、克隆形成、迁移及侵袭能力的影响。研究结果1.QPCR方法检测FADS2在食ESCC及其相对应的癌旁正常粘膜组织中的表达,统计学结果显示FADS2 m RNA在ESCC组织中呈现高表达现象,而在癌旁组织中呈现相对低表达现象,且在ESCC组织和癌旁组织中的表达水平差异具有统计学意义(p0.05)。2.IHC结果提示在有效的208例检测样本中,在癌组织中112例FADS2蛋白阳性表达(53.8%),96例FADS2蛋白阴性表达(46.2%);208例相对应的癌旁正常组织中,63例FADS2蛋白阳性表达(30.3%),145例FADS2蛋白阴性表达(69.7%),FADS2的阳性表达率之间差异有统计学意义(p0.05)。通过统计学分析,发现FADS2高表达与患者淋巴结转移和临床分期有关(P0.05),但与患者年龄、性别、肿瘤分化程度及浸润均未发现有明显相关性(P0.05)。Kaplan-Meier分析结果表明,FADS2高表达组(n=79)与正常表达组(n=129)的生存曲线有显著差异,FADS2高表达患者的中位生存时间18个月,FADS2正常表达患者的中位生存时间27个月,二者差异有统计学意义(P0.05)。3.通过体外细胞功能实验,我们发现过表达FADS2基因的食管癌细胞系KYSE30和KYSE510的增殖、非锚定克隆形成能力增强;同时,细胞迁移实验和侵袭实验显示FADS2能够促进细胞的迁移和侵袭。结论1.FADS2在食管鳞癌组织中呈现阳性表达,且在癌组织中的表达量高于相对应的癌旁组织,其高表达与患者的淋巴结转移及临床分期有关,是预后不良的重要因素。2.FADS2的高表达会导致肿瘤细胞的增殖、迁移、侵袭和非锚定克隆形成能力增强。
[Abstract]:Research background and objective: esophageal cancer is one of the most common malignant tumor of digestive tract, the mortality rate ranked fourth in all [1] tumors, pathological type was squamous cell carcinoma and adenocarcinoma, in esophageal squamous cell carcinoma in China (Esophageal squamous cell carcinoma, ESCC) is the main pathological types, its incidence has the phenomenon of regional differences the obvious and familial aggregation, the high incidence area is located in East Asia and Eastern China, especially Henan, Linzhou, Anyang, Huixian and Dabie Mountain and other places. Oncomolecularbiology current researches show that the environment, genetic and psychological factors such as through synergistic or sequential manner to cause DNA damage, tumor suppressor gene inactivation or activation of oncogene, abnormal changes and apoptosis regulating gene or DNA repair genes, promote normal esophageal mucosa after a multi factor, multi gene, multi stage malignant evolution This process, for the development of esophageal carcinoma [2], accordingly, from the DNA repair gene, oncogene, tumor suppressor gene, genetic polymorphism of metabolic enzyme gene with susceptibility gene and tumor marker research for early diagnosis of esophageal cancer, [3] provides more theoretical basis for treatment and prognosis. The study found that in breast cancer tissue fatty acid desaturase gene 2 (Fatty Acid 2 Desaturase, FADS2) expression, promote linoleic acid (linoleic acid, LA) into twenty four carbon acid (arachidonic acid, AA), and its metabolite of prostaglandin E2 (Prostagland E2 PGE2) increased in breast cancer to to promote the role of [4] in the development of this gene was up-regulated in esophageal squamous cell carcinoma, its mechanism is not clear. Through the experiment, the expression of FADS2 gene was detected in ESCC and adjacent normal mucosa tissue mRNA and protein, To analyze the relationship between clinicopathological characteristics and prognosis of patients; construct FADS2 cell clones stably expressing the lentiviral transfection system, experiments were carried out in vitro cell proliferation, soft agar colony formation assay, colony formation assay, invasion and migration of cells to investigate its biological function. Methods 1. clinical specimens were collected from surgical resection ESCC fresh tissue samples from Linzhou City, Henan province people's Hospital, every specimen containing esophageal squamous cell carcinoma and paracancerous tissue, a total of 70, stored in liquid nitrogen, for the extraction of RNA, the expression of FADS2 mRNA. In addition, the research group of the transfer of the Linzhou City People's Hospital of Henan Province in 299 cases of ESCC radical resection paraffin embedded tissue samples, finishing the clinical pathology and follow-up data,.2. method of tissue microarray of ESCC detected by QPCR FADS2 and ESCC mRNA in 70 cases of carcinoma adjacent to normal phase The expression of mucosal tissues; was detected by FADS2 immunohistochemical method in 299 ESCC tissue microarray expression levels, and to analyze the correlation between FADS2 expression and clinicopathological characteristics and prognosis of ESCC patients. The lentiviral packaging system FADS2 expression plasmids were transfected into KYSE30 and KYSE510 in esophageal carcinoma cell lines. Select the stable expression of FADS2 cells, and QPCR and Western blot. Through the identification of cell proliferation in vitro, colony formation assay, soft agar colony formation assay, migration assay and invasion assay of FADS2 on cell growth, colony formation, migration and invasion effect. The results of 1.QPCR method to detect the expression of FADS2 in food ESCC and adjacent normal mucosa tissues, the statistical results show that the FADS2 m RNA in ESCC tissues showed high expression, while in the adjacent tissues The relatively low expression, statistically significant differences in the level of organization and expression in ESCC tissues and adjacent to the (P0.05).2.IHC results in 208 cases of samples effectively, in cancer tissues of 112 cases of FADS2 protein positive expression (53.8%), 96 cases of negative expression of FADS2 protein (46.2%); 208 cases. The corresponding adjacent normal tissues, 63 cases of positive expression of FADS2 protein (30.3%), 145 cases of negative expression of FADS2 protein (69.7%), there was significant difference between the positive expression rate of FADS2 (P0.05). By statistical analysis, found that the high expression of FADS2 in patients with lymph node metastasis and clinical stage (P0.05), but with age, gender, tumor differentiation and invasion were not found to have significant correlation (P0.05).Kaplan-Meier analysis showed that FADS2 high expression group (n=79) and normal group (n=129) and the expression of the survival curves were significantly different in patients with high FADS2 expression in The survival time of 18 months, FADS2 normal expression of the median survival time of patients 27 months, there was significant difference between the two groups (P0.05).3. cell function by in vitro experiments, we found that overexpression of FADS2 gene in esophageal cancer cell lines KYSE30 and KYSE510 proliferation, anchorage clone formation ability enhancement; at the same time, cell migration assay and invasion assay showed that FADS2 could promote cell migration and invasion. Conclusion 1.FADS2 showed positive expression in esophageal squamous cell carcinoma, and the expression in cancer tissues was higher than that of corresponding adjacent tissues, the high expression of staging and lymph node metastasis and clinical patients, high expression of.2.FADS2 important factors of poor prognosis the cause of tumor cell proliferation, migration, invasion and non anchored clone formation ability.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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