【作者】 熊鹏；

【导师】 朱珉；

【作者基本信息】 华中科技大学 ， 外科学， 2017， 硕士

【摘要】 目的:探讨利用生物信息学方法分析GPR34基因和蛋白在非小细胞肺癌（non-small cell lung cancer,NSCLC）组织及正常肺组织中的差异表达,预测分析GPR34与NSCLC预后的关系;探讨利用生物学实验分析NSCLC GPR34基因表达情况,分析GPR34与NSCLC的相关性;探讨利用基因过表达技术研究NSCLC差异表达基因GPR34对NSCLC细胞增殖、侵袭和迁移的影响。方法:1.联合应用Oncomine、TCGA、HPA数据库分析GPR34基因和蛋白分别在正常肺组织及NSCLC肿瘤组织中的表达差异。同时,应用Kaplan-Meier数据库生存模型预测分析GPR34基因表达与NSCLC患者预后的关系。2.通过实时荧光定量聚合酶链式反应（qRT-PCR）技术检测70例NSCLC肿瘤组织及其配对的癌旁正常组织中GPR34的表达水平,比较癌组织和正常组织之间GPR34的表达差异,采用2^-ΔΔCT法分析GPR34的相对表达倍数,并分析肿瘤组织中GPR34表达水平与临床病理特征之间的关系。3.通过免疫组织化学技术（Immunohistochemistry,IHC）检测70例NSCLC肿瘤组织及癌旁正常组织石蜡切片标本中GPR34、Ki67蛋白表达,分析GPR34、Ki67蛋白表达与临床病理特征之间的关系,并分析GPR34与Ki67蛋白表达的相关性。4.通过qRT-PCR和Western blot技术检测多种NSCLC细胞系中GPR34基因和蛋白表达水平。5.针对GPR34基因设计GPR34过表达载体,转染肺腺癌A549细胞系,使用qRT-PCR检测GPR34 mRNA表达;通过CCK8和Transwell法分别检测细胞增殖、侵袭和迁移。结果:1、联合多个数据库初步表明GPR34基因和蛋白在NSCLC肿瘤组织中下调表达,且其低表达与NSCLC患者（特别是腺癌患者）不良预后密切相关。2、NSCLC肿瘤组织中GPR34 mRNA表达水平为正常肺组织的0.46+0.24倍,显著低于正常肺组织,且GPR34的下调表达与肿瘤分化程度相关。3、GPR34蛋白在正常肺组织高表达,而在NSCLC肿瘤组织低表达,且其表达与肿瘤分化程度显著相关;Ki67蛋白在NSCLC肿瘤组织高表达,且其表达与分化程度显著相关;GPR34与Ki67在NSCLC肿瘤组织中的表达呈一定程度的负相关。4、qRT-PCR和Western blot结果表明,GPR34基因和蛋白在多种NSCLC细胞系中表达下调。5、GPR34特异性过表达载体转染A549细胞后,qRT-PCR检测证实GPR34基因明显高表达;GPR34基因过表达使A549细胞增殖、侵袭和迁移能力减弱。结论:1、探讨性应用生物信息学方法分析NSCLC GPR34基因表达,GPR34在NSCLC中下调表达。2、首次证实GPR34是NSCLC相关基因,其下调表达与NSCLC分化程度、不良预后密切相关。3、GPR34特异性过表达载体转染肺腺癌A549细胞可有效促进GPR34基因表达;GPR34基因过表达后抑制A549细胞增殖、侵袭和迁移。更多还原

【Abstract】 Objective: This study is to investigate the use of bioinformatic methods to analyze the differential expression of GPR34 gene and protein in normal lung tissue and non-small cell lung cancer(NSCLC)tissue,to analyze the relationship between GPR34 and NSCLC prognosis;This study is to investigate GPR34 gene expression by biological experiments,to analyze the correlation of GPR34 and NSCLC;This study is to stimulate the GPR34 gene expression by gene overexpression to investigate the changes in cell growth,invasion and migration.Methods:1、Combined with analysis of Oncomine,TCGA and HPA,the expression levels of GPR34 gene and protein in normal and NSCLC tissues were systematically investigated.In addition,we also analyzed the correlation of GPR34 gene expression and NSCLC prognosis by the survival model of Kaplan-Meier database.2、The expression of GPR34 mRNA was examined by qRT-PCR in 70 paired NSCLC tumor tissues and its matched adjacent normal tissues.We compared the differential expression between them,and further analyzed the correlation of GPR34 and clinicopathologic characteristics.3 、 The expression of GPR34 and Ki67 protein were examined by immunohistochemistry in NSCLC tumor tissues of 70 cases and adjacent normal lung tissues of 70 cases;We analyzed the correlation of GPR34,Ki67 and clinicpathologic characteristics respectively,and further discussed the relationship between GPR34 and Ki67.4、The expression of GPR34 gene and protein in a variety of NSCLC cell lines was detected by qRT-PCR and Western blot.5、The GPR34 overexpression vector was designed and transfected into lung adenocarcinoma A549 cell line.The expression of GPR34 mRNA was detected by qRT-PCR.Cell proliferation was detected by CCK8.Cell invasion and migration were detected by Transwell.Results:1、GPR34 gene and protein were found to be down-regulated in NSCLC by in silico analysis,and its low-expression correlated with poor prognosis in patients with NSCLC,especially lung adenocarcinoma.2、The expression of GPR34 mRNA in NSCLC tissues were 0.46+0.24 times lower than that in normal lung tissues.The expression of GPR34 was related with tumor differentiation of NSCLC.3、GPR34 protein was found to be down-regulated in NSCLC tissues whereas up-regulated in normal lung tissues.Ki67 protein was found to be up-regulated in NSCLC tissues whereas down-regulated in normal lung tissues.The expression of GPR34 was related with tumor differentiation.Ki67 expression was correlated with tumor differentiation.A certain degree of negative correlation exists between GPR34 and Ki67 expression in NSCLC tissues.4、Combined with the results of qRT-PCR and Western blot,GPR34 gene and protein were found to be down-regulated in several NSCLC cell lines.5、After transfection of GPR34 overexpression vector into lung adenocarcinoma A549 cell line,GPR34 gene expression was significantly up-regulated as detected by qRT-PCR.The overexpression of GPR34 gene decreases proliferation,invasion and migration ability of A549 cell.Conclusion:1、The expression of GPR34 gene in NSCLC was analyzed by bioinformatics metheds,and the results indicated that GPR34 gene was down-regulated in NSCLC.2、It was confirmed that GPR34 was the NSCLC-related gene for the first time,and its down-regulated expression was closely related to the NSCLC differentiation and poor prognosis3、GPR34-specific overexpression vector may efficiently up-regulate the expression of GPR34 gene after transfected into A549 cell.GPR34 gene overexpression inhibits the proliferation,invasion and migration ability of A549 cell.更多还原