【作者】 杨阳；

【导师】 董稚明； 郭炜；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2021， 硕士

【摘要】 目的:检测长链非编码RNA(long non-coding RNA,lnc RNA)FOXD3-AS1和FOXD3基因在贲门腺癌(gastric cardia adenocarcinoma,GCA)组织及胃癌细胞系中的表达,分析其与临床病理参数之间的关系;通过体外实验,探讨FOXD3-AS1和FOXD3基因对胃癌细胞增殖,迁移和侵袭等生物学行为的影响及可能的机制。方法:1.通过检索UCSC数据库,分析FOXD3-AS1和FOXD3位置关系。2.应用实时荧光定量反转录多聚核苷酸酶链式反应(Quantitative real-time reverse transcription polymerase chain reaction,q RT-PCR)的方法检测FOXD3-AS1和FOXD3在53例贲门腺癌组织及其癌旁正常组织和四株人胃癌细胞系(SGC-7901、BGC-823、MGC-803、HGC-27)中的表达情况。3.分别构建FOXD3-AS1敲低质粒、FOXD3-AS1过表达质粒、FOXD3过表达质粒,通过q RT-PCR方法验证转染效率。4.应用细胞增殖实验、克隆形成实验、划痕实验和Transwell小室侵袭实验检测FOXD3-AS1和FOXD3在体外对胃癌细胞增殖、迁移及侵袭能力的影响。5.应用q RT-PCR和蛋白质印迹法(western blot)检测FOXD3-AS1和FOXD3对上皮-间质转化(epithelial mesenchymal transition,EMT)相关因子(E-cadherin、N-cadherin、Vimentin、β-catenin)m RNA和蛋白表达水平的影响。6.应用q RT-PCR及蛋白质免疫印迹的方法检测FOXD3-AS1和FOXD3在RNA及蛋白质表达水平上的调节关系。结果:1.UCSC数据库提示,FOXD3-AS1和FOXD3在基因中的位置具有相关性。2.FOXD3-AS1基因在胃癌细胞系SGC-7901、BGC-823、MGC-803、HGC-27中的表达均高于正常细胞对照组(P<0.05);贲门腺癌组织中的FOXD3-AS1表达量明显高于癌旁正常组织(Z=-2.695,P=0.007),并且FOXD3-AS1的表达水平与GCA患者临床分期(Z=-2.981,P=0.003)和淋巴结转移(Z=-3.43,P=0.001)相关。3.FOXD3在胃癌细胞系SGC-7901、BGC-823、MGC-803、HGC-27中的表达均低于正常细胞对照组(P<0.05);贲门腺癌组织FOXD3表达量明显低于癌旁正常组织(Z=-7.649,P<0.01),并且FOXD3的表达水平与GCA患者临床分期(Z=-2.853,P=0.004)和淋巴结转移(Z=-3.187,P=0.001)相关。4.敲低和过表达FOXD3-AS1细胞系的构建及转染效率的验证:与对照组相比,转染si-FOXD3-AS1的SGC-7901细胞中FOXD3-AS1的表达水平明显下调,转染pc DNA3.1-FOXD3-AS1质粒的SGC-7901细胞中FOXD3的表达水平明显上调(P<0.05)。5.过表达FOXD3基因细胞系的构建及转染效率的验证:与对照组相比,转染pc DNA3.1-FOXD3质粒的SGC-7901细胞中FOXD3的表达水平明显上调(P<0.05)。6.细胞增殖实验和平板克隆形成实验结果显示,FOXD3-AS1敲低抑制胃癌细胞SGC-7901的体外增殖能力,FOXD3-AS1过表达增强胃癌细胞SGC-7901的体外增殖能力;划痕愈合实验结果显示,FOXD3-AS1敲低抑制胃癌细胞SGC-7901的迁移能力,FOXD3-AS1过表达增强胃癌细胞SGC-7901的体外迁移能力;Transwell小室侵袭实验结果显示,FOXD3-AS1敲低抑制胃癌细胞SGC-7901的侵袭能力,FOXD3-AS1过表达增强胃癌细胞SGC-7901的体外侵袭能力;FOXD3-AS1敲低可上调胃癌细胞SGC-7901中E-cadherin的m RNA表达水平,降低N-cadherin、β-catenin和vimentin的m RNA表达水平,过表达则作用相反;FOXD3-AS1敲低可上调胃癌细胞SGC-7901中E-cadherin的蛋白表达水平,降低N-cadherin、β-catenin和vimentin的蛋白表达水平,过表达则作用相反(P<0.05)。7.细胞增殖实验和平板克隆形成实验结果显示,FOXD3过表达会抑制胃癌细胞SGC-7901的体外增殖能力;划痕愈合实验结果显示,FOXD3过表达会抑制胃癌细胞SGC-7901的迁移能力;Transwell小室侵袭实验结果显示,FOXD3过表达会抑制胃癌细胞SGC-7901的侵袭能力;FOXD3过表达可上调胃癌细胞SGC-7901中E-cadherin的m RNA表达水平,降低N-cadherin、β-catenin和vimentin的m RNA表达水平;FOXD3过表达可上调胃癌细胞SGC-7901中E-cadherin的蛋白表达水平,降低N-cadherin、β-catenin和vimentin的蛋白表达水平(P<0.05)。8.构建敲低和过表达FOXD3-AS1基因的SGC-7901细胞系,q RT-PCR检测FOXD3的表达水平:FOXD3基因的相对表达量在FOXD3-AS1敲低组显著高于对照组;FOXD3基因的相对表达量在FOXD3-AS1过表达组显著低于对照组。9.利用Western blot检测FOXD3-AS1敲低和过表达后胃癌细胞系SGC-7901中FOXD3的蛋白表达:在敲低FOXD3-AS1的表达后,胃癌细胞中FOXD3的蛋白表达水平均明显升高;在过表达FOXD3-AS1后,胃癌细胞中FOXD3的蛋白表达水平明显降低。结论:1.FOXD3-AS1在贲门腺癌组织和胃癌细胞系中的表达明显上调,FOXD3在贲门腺癌组织和胃癌细胞系中的表达明显下调,且都与患者的临床分期和淋巴结转移密切相关。2.FOXD3-AS1敲低会抑制胃癌细胞的体外增殖,迁移与侵袭能力,FOXD3-AS1过表达会增强胃癌细胞的体外增殖,迁移与侵袭能力,FOXD3-AS1可能通过参与EMT通路相关因子的表达影响胃癌细胞的侵袭和转移进程。3.FOXD3过表达会抑制胃癌细胞的体外增殖,迁移与侵袭能力,FOXD3可能通过参与EMT通路相关因子的表达影响胃癌细胞的侵袭和转移进程。4.FOXD3-AS1在转录及转录后水平对FOXD3的表达具有负向调节作用。更多还原

【Abstract】 Objective:To detect the expression of lnc RNA FOXD3-AS1 and FOXD3 gene in gastric cardia adenocarcinoma(GCA)tissues and gastric cancer cell lines,and to explore the relationship between expression level and clinicopathological parameter.The effects of FOXD3-AS1 and FOXD3 genes on the proliferation,migration and invasion of gastric cancer cells were investigated in vitro experiments.Methods:1.By searching UCSC database,the position relationship between FOXD3-AS1 and FOXD3 was analyzed.2.q RT-PCR was used to detect the expression level of FOXD3-AS1 and FOXD3 in 53 cases gastric cardia adenocarcinoma tissues and normal tissue adjacent to carcinoma and four human gastric cancer cell line(SGC-7901 and BGC-823,MGC-803,the HGC-27).3.FOXD3-AS1 knockdown and overexpression plasmids were constructed,and FOXD3 overexpression plasmids were synthesized,the transfection efficiency was verified by q RT-PCR.4.The effects of FOXD3-AS1 and FOXD3 on the proliferation,migration and invasion of gastric cancer cells in vitro were detected by cell proliferation assay,clone formation assay,scratch assay and Transwell chamber invasion assay.5.The effects of FOXD3-AS1 and FOXD3 on the m RNA and protein expression levels of Epithelial mesenchymal transition(EMT)related factors(E-cadherin,N-cadherin,Vimentin,β-catenin)were detected by q RT-PCR and Western blot.6.The regulate relationship of FOXD3-AS1 and FOXD3 on RNA and protein expression levels was detected by q RT-PCR and Western blot.Results:1.UCSC database suggested that FOXD3-AS1 and FOXD3 were correlated in gene positions.2.The expression of FOXD3-AS1 gene in gastric cancer cell lines SGC-7901,BGC-823,MGC-803 and HGC-27 were higher than those in normal control group(P<0.05);the expression of FOXD3-AS1 in gastric cardia adenocarcinoma tissues was significantly higher than that in adjacent normal tissues(Z=-2.695,P=0.007),and the expression level of FOXD3-AS1 was correlated with clinical stage(Z=-2.981,P=0.003)and lymph node metastasis(Z=-3.43,P=0.001)of GCA patients.3.The expressions of FOXD3 in gastric cancer cell lines SGC-7901,BGC-823,MGC-803 and HGC-27 were lower than those in normal control group(P<0.05);the expression of FOXD3 in cardia adenocarcinoma was significantly lower than that in adjacent normal tissues(Z=-7.649,P<0.01),and the expression of FOXD3 was correlated with the clinical stage(Z=-2.853,P=0.004)and lymph node metastasis(Z=-3.187,P=0.001)of GCA patients.4.Construction of knockdown and overexpression of FOXD3-AS1 cell lines and verification of transfection efficiency: compared with the control group,the expression level of FOXD3-AS1 in SGC-7901 cells transfected with si-FOXD3-AS1 was significantly down-regulated,and the expression level of FOXD3 in SGC-7901 cells transfected with pc DNA3.1-FOXD3-AS1 plasmid was significantly up-regulated(P<0.05).5.Construction of overexpressed FOXD3 cell lines and verification of transfection efficiency: compared with the control group,the expression level of FOXD3 in SGC-7901 cells transfected with pc DNA3.1-FOXD3 plasmid was significantly up-regulated(P<0.05).6.The results of cell proliferation assay and plate clone formation assay showed that FOXD3-AS1 knockdown inhibited the proliferation ability of gastric cancer cells SGC-7901 in vitro,while FOXD3-AS1 overexpression enhanced the proliferation ability of gastric cancer cells SGC-7901 in vitro.The results of scratch healing assay showed that FOXD3-AS1 knockdown inhibited the migration ability of gastric cancer cells SGC-7901,while FOXD3-AS1 overexpression enhanced the migration ability of gastric cancer cells SGC-7901 in vitro.Transwell compartment invasion assay showed that FOXD3-AS1 knockdown inhibited the invasion ability of gastric cancer cells SGC-7901,while FOXD3-AS1 overexpression enhanced the invasion ability of gastric cancer cells SGC-7901 in vitro.FOXD3-AS1 knockdown can up-regulate the m RNA expression of E-cadherin in gastric cancer cells SGC-7901,and decrease the m RNA expression of N-cadherin,β-catenin and vimentin,while overexpression has the opposite effect.FOXD3-AS1 knockdown could up-regulate the protein expression levels of E-cadherin in SGC-7901,and decrease the protein expression levels of N-cadherin,β-catenin and vimentin,while over-expression had the opposite effect(P <0.05).7.The results of cell proliferation assay and plate clone formation assay showed that FOXD3 overexpression could inhibit the proliferation of gastric cancer cell line SGC-7901 in vitro.The results of scratch healing assay showed that FOXD3 overexpression could inhibit the migration of gastric cancer cell line SGC-7901.Transwell compartment invasion assay showed that Fox D3 overexpression inhibited the invasion ability of SGC-7901.FOXD3 overexpression can up-regulate the m RNA expression of E-cadherin in SGC-7901,and decrease the m RNA expression of N-cadherin,β-catenin and vimentin.FOXD3 overexpression up-regulated the protein expression level of E-cadherin in SGC-7901,and decreased the protein expression levels of N-cadherin,β-catenin and vimentin(P < 0.05).8.SGC-7901 cell lines with knockdown and overexpression of FOXD3-AS1 gene were constructed to detect the expression level of FOXD3 by q RT-PCR: The relative expression level of FOXD3 gene in the knockdown group was significantly higher than that in the control group;the relative expression of FOXD3 gene in FOXD3-AS1 overexpression group was significantly lower than that in control group.9.Western blot was used to detect the protein expression of FOXD3 in gastric cancer cell line SGC-7901 after knockdown and overexpression of FOXD3-AS1: the expression level of FOXD3 protein in gastric cancer cells was significantly increased after the knockdown of FOXD3-AS1;the expression level of FOXD3 protein in gastric cancer cells was significantly decreased after the overexpression of FOXD3-AS1.Conclusion:1.The expression of FOXD3-AS1 was significantly up-regulated in GCA tissues and gastric cancer cell lines,and the expression of FOXD3 was significantly down-regulated in GCA tissues and gastric cancer cell lines,both of which were closely related to the clinical stage and lymph node metastasis of patients.2.FOXD3-AS1 knockdown can inhibit the proliferation,migration and invasion ability of gastric cancer cells in vitro,and the overexpression of FOXD3-AS1 can enhance the proliferation,migration and invasion ability of gastric cancer cells in vitro.FOXD3-AS1 may affect the invasion and metastasis process of gastric cancer cells by participating in the expression of EMT pathway related factors.3.FOXD3 overexpression can inhibit the proliferation,migration and invasion ability of gastric cancer cells in vitro,and FOXD3 may affect the invasion and metastasis process of gastric cancer cells by participating in the expression of EMT pathway related factors.4.FOXD3-AS1 negatively regulates the expression of FOXD3 at the transcriptional and post-transcriptional levels.更多还原