【作者】 马晓丽；

【导师】 张莉；

【作者基本信息】 新疆医科大学 ， 肿瘤学（专业学位）， 2021， 博士

【摘要】 目的:本研究目的是探讨整合素连接激酶(ILK)基因对食管癌细胞增殖、凋亡、侵袭和迁移的影响,研究其在食管癌中功能,为食管癌的精准诊疗奠定理论基础和临床参考。方法:1)采用meta分析评价ILK在消化系统肿瘤组织中的表达,及与消化系统肿瘤临床表型及预后间的相关性;2)筛选ILK高表达的人食管鳞癌细胞系,建立ILK慢病毒干扰序列。将ILK慢病毒干扰序列转染食管鳞癌细胞系TE-1和KYSE-150,用q RT-PCR检测干扰载体转染后ILK的表达。在细胞水平上,利用MTT增殖、克隆检测、流式细胞仪凋亡检测、Transwell小室侵袭及划痕愈合等技术,研究ILK基因消减对食管鳞癌细胞增殖、凋亡、侵袭和迁移的影响;3)建立ILK慢病毒干扰载体,转染KYSE-150细胞。在裸鼠皮下分别接种sh ILK及对照细胞株,建立KYSE-150细胞裸鼠皮下移植瘤模型,通过观察肿瘤生长,测量肿瘤大小和体积。在动物水平上,研究ILK基因消减对食管鳞癌细胞生长的影响。基于RNA-seq技术筛选食管癌的差异表达基因,通过GO功能富集和KEGG通路分析预测其可能的发病机制,筛选可能参与食管癌发生发展的关键调控基因及相关信号通路。结果:第一部分:meta分析结果显示ILK在消化系统肿瘤组中高表达,ILK表达与肿瘤分化程度、TNM分期、淋巴结转移、肿瘤浸润程度及生存预后呈相关性,与年龄及性别无明显相关性。第二部分:在体外ESCC细胞水平,沉默ILK基因后,可显著抑制TE-1、KYSE-150细胞增殖和集落形成,促进TE-1、KYSE-150细胞凋亡。ILK基因消减后,TE-1、KYSE-150细胞侵袭及迁移能力明显减弱。第三部分:裸鼠移植瘤动物模型结果发现,ILK干扰组裸鼠肿瘤生长缓慢,相比较于对照组,肿瘤的重量及体积均偏小,ILK基因消减后影响了裸鼠成瘤的能力。基于RNA-seq技术,ILK基因干扰后挖掘到5540个食管癌差异表达基因,其中上调基因为2839个,下调基因为2601个。上调的基因有SOD2、IRF6、PER1、PHLDB2、TNFAIP3等,下调的基因有RPL21、HYOU1、WARS、SUPT16H、DHCR24等。可能参与TNF信号通路、AMPK信号通路、Fox O信号通路、P53信号通路、NF-k B信号通路等。结论:ILK在消化系统肿瘤中高表达,与肿瘤分化程度、TNM分期、淋巴结转移、肿瘤浸润程度及生存预后呈相关性,与年龄及性别无明显相关性。ILK基因消减抑制ESCC细胞增殖、克隆形成、侵袭及转移,促进细胞凋亡。体内致瘤研究证实,ILK基因消减会阻碍KYSE-150移植瘤的生长。基于RNA-seq技术筛选出食管癌的差异表达基因,通过GO功能富集和KEGG通路分析预测差异表达基因,可能相关的信号传导通路。更多还原

【Abstract】 Objective: The purpose of this study was to investigate the effect of integrin-linked kinase(ILK)gene on the proliferation,apoptosis,invasion and migration of esophageal cancer cells,and to study its function in esophageal cancer,so as to lay a theoretical foundation and clinical reference for accurate diagnosis and treatment of esophageal cancer.Methods: 1)meta analysis was used to evaluate the expression of ILK in digestive system tumors and its correlation with clinical phenotype and prognosis of digestive system tumors.2)Human esophageal squamous cell carcinoma lines with high expression of ILK were screened to establish ILK lentivirus interference sequence.The ILK lentivirus interference sequence was transfected into esophageal squamous cell carcinoma cell lines TE-1 and KYSE-150,The expression of ILK after transfection was detected by q RT-PCR.At the cellular level,MTT proliferation,clone detection,flow cytometry apoptosis detection,Transwell chamber invasion and scratch healing were used to study the effects of ILK gene deletion on the proliferation,apoptosis,invasion and migration of esophageal squamous cell carcinoma cells.3)establish ILK lentivirus interference vector and transfect KYSE-150 cells.Sh ILK and control cell lines were inoculated subcutaneously in nude mice to establish the subcutaneous transplanted tumor model of KYSE-150 cells in nude mice.The tumor growth was observed and the size and volume of the tumor were measured.To study the effect of ILK gene deletion on the growth of esophageal squamous cell carcinoma at animal level.The differentially expressed genes of esophageal cancer were screened based on RNA-seq.The possible pathogenesis of esophageal cancer was predicted through GO function enrichment and KEGG pathway analysis,and the key regulatory genes and related signal pathways that may be involved in the occurrence and development of esophageal cancer were screened.Results: Part1: the results of meta analysis showed that ILK was highly expressed in digestive system tumors.The expression of ILK was correlated with tumor differentiation,TNM stage,lymph node metastasis,tumor invasion and survival prognosis,but not with age and sex.Part 2: at the level of ESCC cells in vitro,silencing ILK gene could significantly inhibit the proliferation and colony formation of TE-1 and KYSE-150 cells,and promote the apoptosis of TE-1 and KYSE-150 cells.After the deletion of ILK gene,the invasion and migration ability of TE-1 and KYSE-150 cells decreased significantly.Part3:the results of the animal model of transplanted tumor in nude mice showed that the tumor in the ILK interference group grew slowly,and the weight and volume of the tumor were smaller than those in the control group.The subtraction of ILK gene affected the ability of tumor formation in nude mice.Based on RNA-seq technique,5540 differentially expressed genes in esophageal cancer were found after ILK gene interference,including 2839up-regulated genes and 2601 down-regulated genes.The up-regulated genes are SOD2,IRF6,PER1,PHLDB2,TNFAIP3,etc.while the down-regulated genes are RPL21,HYOU1,WARS,SUPT16 H,DHCR24 and so on.It may be involved in TNF signal pathway,AMPK signal pathway,Fox O signal pathway,p53 signal pathway,NF-k B signal pathway and so on.Conclusion: The high expression of ILK in digestive system tumors was correlated with tumor differentiation,TNM stage,lymph node metastasis,tumor invasion and survival prognosis,but not with age and sex.ILK gene deletion inhibits the proliferation,clone formation,invasion and metastasis of ESCC cells,and promotes apoptosis.In vivo tumorigenesis studies have confirmed that the deletion of ILK gene will hinder the growth of KYSE-150 transplanted tumor.The differentially expressed genes in esophageal cancer were screened based on RNA-seq technique,and the differentially expressed genes and possible related signal transduction pathways were predicted by GO functional enrichment and KEGG pathway analysis.更多还原