【作者】 李鑫；

【导师】 沈孝兵；

【作者基本信息】 东南大学 ， 劳动卫生与环境卫生学， 2021， 硕士

【摘要】 背景:胃癌的发病率和致死率分居世界第五和第四,严重威胁公众的生命健康。此外胃癌五年生存率低,预后往往不良,因此寻找一个胃癌的独立预后因子意义重大。Lumican(LUM)是小富亮氨酸蛋白聚糖(SLRPs)家族中重要的一类,其对肿瘤的发生、发展存在着两种不同的作用,它既可能是癌基因,也可能是抑癌基因,关于lumican对肿瘤的作用目前仍然存在争议。本研究着重探索了lumican在胃癌中表达的临床意义及其分子机制,为胃癌预后标志物的筛选以及靶点治疗提供更多的依据。目的:1.基于公共数据库TCGA,结合qRT-PCR实验和meta分析,探讨lumican在胃癌中的表达及其预后意义。2.研究lumican对胃癌细胞MGC-803、AGS的增殖、迁移和侵袭能力等生物学功能的影响。3.探究敲低/过表达lumican对胃癌MGC-803、AGS细胞中MAPK信号转导通路的影响。4.研究ERK抑制剂GDC-0994对过表达lumican的胃癌细胞MGC-803、AGS中MAPK信号转导通路的影响,初步探讨lumican的促胃癌机制,为胃癌的靶点治疗提供更多的实验依据。方法:1.Lumican表达与临床病理参数之间的相关性及预后意义。胃癌的基因表达数据和临床数据来自于癌症基因组图谱(TCGA)数据库。通过perl语言对数据进行筛选和整理后,使用R软件分析lumican在胃癌组织和正常组织中的表达差异,并采用实时荧光定量聚合酶链式反应(qRT-PCR)和meta分析进行验证。Lumican表达与临床病理参数之间的关系通过卡方检验和Logistic回归进行分析。利用Kaplan-Meier生存曲线和Cox比例风险回归模型来评估lumican表达与总生存之间的相关性。基因集富集分析(GSEA)用于筛选高低表达lumican表型显著富集的胃癌相关信号传导途径。2.明确lumican表达变化对胃癌细胞生物学功能的影响。首先对不同胃癌细胞株进行m RNA和蛋白水平的检测和验证,并筛选出MGC-803和AGS细胞用于后续实验。分别通过si RNA和慢病毒载体建立lumican敲低和过表达胃癌细胞模型,并在m RNA水平和蛋白水平检测转染效率。利用CCK-8细胞增殖实验、细胞划痕愈合实验以及transwell迁移侵袭等功能实验探索敲低/过表达对胃癌细胞生物学功能的影响。3.探索lumican影响胃癌发生发展的分子机制。利用Western Blot检测敲低/过表达lumican的MGC-803和AGS细胞中MAPK信号通路关键分子的表达变化。进一步探究ERK分子抑制剂GDC-0994是否回复了由于lumican过表达所致MGC-803和AGS细胞中MAPK信号通路关键分子改变。结果:1.TCGA数据库中lumican基因在胃癌组织中的表达水平显著高于正常组织(P<0.001)。qRT-PCR(P<0.01)和meta分析(SMD=0.90,95%CI:0.34-1.46)进一步验证了lumican在胃癌组织中表达上调。卡方检验显示lumican的高表达与肿瘤分化程度(P=0.024)和T分期(P=0.004)相关。Logistic回归分析显示lumican的表达水平与肿瘤分化程度(低分化相比较高或中分化,OR=1.543,P=0.043),病理分期(II期相比较I期,OR=3.149,P=0.001;Ⅲ期相比较Ⅰ期,OR=2.505,P=0.007)和T分期(T2期相比较T1期,OR=13.304,P=0.014;T3期相比较T1期,OR=18.434,P=0.005;T4期相比较T1期,OR=30.649,P=0.001)呈显著的正相关性。KaplanMeier曲线表明,lumican高表达的胃癌患者预后较差。多因素Cox分析表明,lumican高表达是胃癌患者预后不良的独立因子(HR=1.189;95%CI,1.011-1.400;P=0.037)。GSEA指出具有高lumican表达表型的样本中富集了14条与癌症相关的信号转导通路,低lumican表达表型的样本中富集了14条正常生理代谢相关分子和信号转导通路。2.qRT-PCR和Western blot检测人胃癌细胞株AGS、MGC-803、MKN45、HGC-27与人正常胃粘膜上皮细胞GES-1中lumican m RNA和蛋白水平的表达。结果显示MGC-803和AGS细胞的lumican m RNA和蛋白水平表达较高,因此筛选出MGC-803和AGS细胞用于后续实验。3.分别利用si RNA和慢病毒载体转染MGC-803和AGS细胞构建敲低/过表达细胞模型。qRT-PCR和Western Blot在m RNA和蛋白水平的检测结果表明敲低/过表达细胞模型成功建立。4.敲低/过表达lumican对胃癌细胞株生物学功能的影响。CCK-8实验结果显示过表达lumican的MGC-803、AGS细胞增殖能力高于对照组。反之,敲低lumican的MGC-803、AGS细胞增殖能力低于对照组。细胞划痕愈合实验结果提示,敲低lumican的MGC-803、AGS细胞划痕愈合较慢,迁移能力减弱,过表达lumican的MGC-803、AGS细胞划痕愈合和迁移能力增强。Transwell迁移侵袭实验结果提示,敲低lumican基因的MGC-803、AGS细胞迁移能力和侵袭能力减弱,反之过表达lumican的MGC-803、AGS细胞迁移能力和侵袭能力增强。5.敲低/过表达lumican的MGC-803、AGS细胞中MAPK信号通路关键信号分子ERK、MEK、p-ERK、p-MEK的表达变化。Western Blot检测结果发现,在敲低lumican的MGC-803、AGS细胞中MEK、p-MEK蛋白水平表达不变,ERK、p-ERK表达减少。过表达lumican的MGC-803、AGS细胞中MEK、p-MEK表达不变,ERK、p-ERK表达上调。6.抑制剂GDC-0994作用于过表达lumican的MGC-803、AGS细胞,Western Blot检测MAPK信号通路关键蛋白分子ERK、MEK、p-ERK、p-MEK的表达变化。结果发现,ERK、p-ERK蛋白水平上的表达回复到对照组水平。结论:1.通过对公共数据库TCGA中的数据分析发现,lumican在胃癌组织中高表达。同时胃癌组织学分级以及病理分期越高,lumican表达越高。生存分析表明,高表达lumican的患者预后更差。Cox回归分析表明lumican是胃癌患者的独立预后因子。此外,高表达lumican的表型中存在多种癌症相关信号通路富集。2.在胃癌细胞株MGC-803和AGS中,lumican m RNA和蛋白水平表达较高。利用si RNA和慢病毒载体转染成功建立敲低/过表达lumican的MGC-803和AGS细胞模型。敲低lumican抑制了MGC-803、AGS细胞增殖能力、迁移和侵袭能力低于对照组。相反,过表达lumican促进了MGC-803、AGS细胞增殖、迁移和侵袭。3.MAPK信号通路的关键分子ERK、p-ERK蛋白表达水平随着Lumican表达改变而发生相应变化。提示lumican可能通过调节MAPK通路来参与胃癌发生发展。更多还原

【Abstract】 Background:The incidence rate and mortality rate of gastric cancer are fifth and fourth respectively,which seriously threaten the public’s health and life.In addition,the fiveyear survival rate of gastric cancer is low and the prognosis is often poor,so it is significant to find an independent prognostic factor for gastric cancer.Lumican(LUM)is an important class of the small leucine-rich proteoglycan(SLRPs)family,which has two different effects on tumorigenesis and development,and it may be both an oncogene and an oncogene suppressor.The role of lumican in tumor is still controversial.This study focused on exploring the clinical significance and molecular mechanism of lumican expression in gastric cancer,providing more basis for screening prognostic markers and targeted therapy of gastric cancer.Objective:1.To explore the expression of lumican in gastric cancer and its prognostic significance based on the public database TCGA,combined with qRT-PCR experiments and meta-analysis.2.To investigate the effect of lumican on biological functions such as proliferation,migration and invasion ability of gastric cancer cells MGC-803 and AGS.3.To investigate the effect of knockdown/overexpression of lumican on MAPK signaling pathway in gastric cancer MGC-803 and AGS cells.4.To study the effect of ERK inhibitor GDC-0994 on MAPK signaling in MGC-803 and AGS cells overexpressing lumican,and to preliminarily explore the mechanism of lumican in promoting cancer,so as to provide more experimental basis for targeted therapy of gastric cancer.Methods:1.Correlation between lumican expression and clinicopathological parameters and prognostic significance.Gene expression profiles and clinical data of gastric cancer(GC)were downloaded from The Cancer Genome Atlas(TCGA)database.After screening and organizing the data by perl language,the differences of lumican expression in gastric cancer tissues and normal tissues were analyzed by R software and validated by quantitative real-time polymerase chain reaction(qRT-PCR)and meta-analysis.The relationship between lumican expression and clinicopathological parameters was analyzed by chi-square test and logistic regression.Kaplan-Meier survival curves and Cox proportional hazards regression models were used to assess the correlation between lumican expression and overall survival.Gene sets enrichment analysis(GSEA)was used to screen for significantly enriched gastric cancer-associated signaling pathways with high and low lumican expression phenotypes.2.To clarify the effect of lumican expression on the biological function of gastric cancer cells.Firstly,different gastric cancer cell lines were examined and validated at m RNA and protein levels,and MGC-803 and AGS cells were screened for subsequent experiments.The lumican knockdown/overexpression gastric cancer cell lines were established by si RNA and lentiviral vectors,respectively,and the transfection efficiency was verified at the m RNA level and protein level.Cell counting kit-8(CCK8)cell proliferation assay,wound healing assay and Transwell migration and invasion assay were used to explore the effect of knockdown/ overexpression of luminican on the biological function of gastric cancer cells.3.To explore the molecular mechanism of lumican affecting the occurrence and development of gastric cancer.Western blot was used to detect the expression of key proteins of MAPK signaling pathway in MGC-803 and AGS cells with knockdown/overexpression of lumican.To further explore whether ERK inhibitor GDC-0994 can restore the changes of key proteins of MAPK signaling pathway in MGC-803 and AGS cells caused by overexpression of lumican.Results:1.The expression level of lumican in gastric cancer was significantly higher than that in normal tissues(P<0.001).Meanwhile,qRT-PCR(P<0.01)and meta-analysis(SMD=0.90,95% CI: 0.34-1.46)further confirmed the up-regulation of lumican expression in gastric cancer.The chi-square test showed that high lumican expression correlated with the degree of tumor differentiation(P=0.024)and T stage(P=0.004).Logistic regression analysis showed that high lumican expression was associated with the degree of tumor differentiation(poor vs.well or moderate,OR=1.543,P=0.043),pathological stage(stage II vs.stage I,OR=3.149,P=0.001;stage III vs.stage I,OR=2.505,P=0.007)and T classification(T2 stage vs.T1 stage,OR=13.043,P=0.004;T2 vs.T1,OR=13.304,P=0.014;T3 vs.T1,OR=18.434,P=0.005;T4 vs.T1,OR=30.649,P=0.001).Kaplan-Meier curves indicated that patients with high lumican expression had a poorer prognosis.Multivariate Cox analysis showed that high expression of lumican was an independent prognostic factor for poor prognosis of gastric cancer patients(HR = 1.189;95%CI,1.011-1.400;P=0.037).GSEA indicated that samples with high lumican expression phenotypes were enriched for 14cancer-related signaling pathways,and samples with low lumican expression phenotypes were enriched for 14 normal physiological metabolism-related molecules and signaling pathways.2.qRT-PCR and Western Blot were performed to detect the expression of lumican m RNA and protein levels in human gastric cancer cell lines AGS,MGC-803,MKN45,HGC-27 and human gastric mucosal epithelial cell line GES-1.The results showed that the expression of lumican m RNA and protein levels were higher in MGC-803 and AGS cells,so MGC-803 and AGS cells were screened for subsequent experiments.3.MGC-803 and AGS cells were transfected with si RNA and lentiviral vectors to construct knockdown/ overexpression cell models,respectively.The results of qRTPCR and Western Blot at m RNA and protein levels indicated that the knockdown/overexpression models were successfully established.4.The effect of knockdown/overexpression of lumican on the biological function of gastric cancer cell lines.The results of CCK-8 assay showed that the proliferation ability of MGC-803 and AGS cells overexpressing lumican was higher than that of the control group.Conversely,the proliferation ability of MGC-803 and AGS cells with lumican knockdown was lower than that of the control group.The results of cell scratch healing assay showed that MGC-803 and AGS cells with knockdown lumican gene had slower scratch healing and weaker migration ability,while MGC-803 and AGS cells with overexpression of lumican had scratch healing and enhanced migration ability.The results of transwell migration and invasion assay showed that the migration and invasion ability of MGC-803 and AGS cells with knockdown of lumican gene were decreased,on the contrary,the migration and invasion ability of MGC-803 and AGS cells with overexpression of lumican gene were enhanced.5.The expression changes of key signaling molecules ERK,MEK,p-ERK,and p-MEK in MAPK signaling pathway in MGC-803 and AGS cells that knockdown/overexpression lumican.Western blot revealed that the expression of MEK and pMEK protein levels were unchanged and the expression of ERK and p-ERK was reduced in the knockdown lumican MGC-803 and AGS cells.The expression of MEK and p-MEK was unchanged in MGC-803 and AGS cells overexpressing lumican,and the expression of ERK and p-ERK was upregulated.6.The inhibitor GDC-0994 acts on MGC-803 and AGS cells overexpressing lumican,and Western Blot was used to detect changes in the expression of ERK,MEK,p-ERK,p-MEK,key protein molecules of MAPK signaling pathway.The results revealed that the expression on the level of ERK and p-ERK proteins reverted to the control level.Conclusions:1.The analysis of data in the public database TCGA revealed that lumican was highly expressed in gastric cancer tissues.At the same time,the higher the histological grade and pathological stage of gastric cancer,the higher the expression of lumican.Survival analysis showed that patients with high expression of lumican had worse prognosis.Cox regression analysis found that lumican was a prognostic factor for gastric cancer.In addition,a variety of cancer-related signaling pathways are enriched in the phenotype with high expression of lumican.2.The expression of lumican m RNA and protein was higher in MGC-803 and AGS cells.MGC-803 and AGS were successfully established by si RNA and lentiviral vector transfection.Knockdown of lumican inhibited the proliferation ability,migration and invasion ability of MGC-803 and AGS cells lower than the control group.In contrast,overexpression of lumican promoted MGC-803,AGS cell proliferation,migration and invasion.3.The expression levels of ERK and p-ERK proteins,key molecules of MAPK signaling pathway,changed accordingly with the alteration of lumican expression.It is suggested that lumican may be involved in the development of gastric carcinogenesis through regulating MAPK pathway.更多还原