【作者】 陈超；

【导师】 朱甲明；

【作者基本信息】 吉林大学 ， 临床医学硕士（外科学）（专业学位）， 2020， 硕士

【摘要】 目的:验证长链非编码RNA(lncRNA)RP11-363E7.4在胃癌组织和癌旁正常组织以及胃癌细胞系中的表达情况,分析患者RP11-363E7.4的表达水平与临床病理特征的相关性,研究RP11-363E7.4过表达后对胃癌细胞系增殖、凋亡、迁移、侵袭能力的影响,探讨其可能潜在的分子机制。方法:1.利用实时荧光定量PCR(qRT-PCR)检测lncRNA RP11-363E7.4在82对胃癌组织和癌旁正常组织中的表达水平,并与患者的临床病理特征进行相关性分析。2.利用qRT-PCR检测lncRNA RP11-363E7.4在3种人胃癌细胞系(SGC-7901,BGC-823,MGC-803)和人正常胃黏膜上皮细胞系(GES-1)中的表达水平。构建lncRNA RP11-363E7.4稳定过表达的胃癌细胞系。利用qRT-PCR检测转染细胞的转染效率。3.通过CCK-8细胞增殖实验、克隆形成实验、流式细胞术、划痕实验、Transwell侵袭实验检测过表达lncRNA RP11-363E7.4对胃癌细胞增殖、凋亡、迁移、侵袭能力的影响。4.通过蛋白质免疫印迹实验(western blot)检测过表达lncRNA RP11-363E7.4的胃癌细胞中凋亡及上皮间充质转化(EMT)相关蛋白的表达水平,探讨lncRNA RP11-363E7.4发挥作用可能的分子机制。结果:1.LncRNA RP11-363E7.4在胃癌组织中的表达水平明显下调(p<0.05)。且lncRNA RP11-363E7.4的表达水平与患者术后病理T分期有一定的相关性(p<0.05),而与年龄、性别、Borrmann分型、肿瘤大小、L分期、N分期不相关(均p>0.05)。2.LncRNA RP11-363E7.4在3种人胃癌细胞系中表达水平均显著低于GES-1,尤其在SGC-7901和MGC-803细胞系中表达水平最低。LncRNA RP11-363E7.4稳定过表达细胞系构建完成后检测转染效率,其在SGC-7901细胞中表达上调32.49倍(p<0.05),在MGC-803细胞中表达上调33.92倍(p<0.05)。3.LncRNA RP11-363E7.4过表达可以抑制胃癌细胞的增殖能力,可以诱导胃癌细胞的凋亡发生。且lncRNA RP11-363E7.4过表达组的凋亡相关蛋白Caspase-3、Bax和p53表达水平上调,Bcl-2表达水平下调。4.LncRNA RP11-363E7.4过表达可以抑制胃癌细胞的迁移和侵袭能力。且lncRNA RP11-363E7.4过表达组的EMT相关蛋白E-cadherin表达水平上调,Ncadherin、Vimentin、Slug和β-catenin表达水平下调。结论:1.长链非编码RNA RP11-363E7.4在胃癌组织和胃癌细胞系中呈低表达水平,且与肿瘤T分期相关。2.长链非编码RNA RP11-363E7.4可以抑制胃癌细胞的增殖、迁移、侵袭能力,可以增强胃癌细胞的凋亡能力,发挥一定的抑癌作用。3.长链非编码RNA RP11-363E7.4可以影响胃癌细胞中凋亡相关蛋白及EMT相关蛋白的表达水平,可能与其在胃癌细胞中发挥抑癌作用有一定的联系。更多还原

【Abstract】 Objective:To investigate the expression level of long non-coding RNA(lncRNA)RP11-363E7.4 in gastric cancer(GC)tissues,adjacent normal tissues and GC cell lines,the correlation between the expression level of lncRNA RP11-363E7.4 and the clinicopathological characteristics of GC patients,the effects of lncRNA RP11-363E7.4 on proliferation,apoptosis,migration and invasion of GC cells,and the underlying potential molecular mechanism.Methods:1.The expression level of lncRNA RP11-363E7.4 in 82 paired of GC tissues and adjacent normal tissues were detected by qRT-PCR.Then the correlation between lncRNA RP11-363E7.4 and the clinicopathological characteristics of GC patients were analyzed.2.The expression level of lncRNA RP11-363E7.4 in 3 human GC cell lines(SGC-7901,BGC-823,MGC-803)and human normal gastric mucosa epithelial cell(GES-1)were detected by qRT-PCR.Followed by the construction of a stable overexpression cell line of lncRNA RP11-363E7.4.Then the transfection efficiency of lncRNA RP11-363E7.4 was detected by qRT-PCR.3.The effects of lncRNA RP11-363E7.4 on the proliferation,apoptosis,migration and invasion of gastric cancer cells were detected by CCK-8 cell proliferation assay,colony formation assay,flow cytometry,scratch wound healing assay and Transwell migration and invasion assay.4.The expression level of proteins related to apoptosis and EMT in lncRNA RP11-363E7.4 overexpressed GC cells were detected by western blot assay to explore the underlying potential molecular mechanisms.Results:1.The expression level of lncRNA RP11-363E7.4 in GC tissues was significantly lower than that in the corresponding normal tissues(p <0.05).The expression level of lncRNA RP11-363E7.4 was correlated with T stage(p<0.05),but not with age,gender,Borrmann classification,tumor size,M stage and N stage(all p>0.05).2.The expression level of lncRNA RP11-363E7.4 in three human GC cell lines was significantly lower than that in GES-1 cell line,and the expression level in SGC-7901 and MGC-803 cell lines were lower than that in BGC-823 cell line.LncRNA RP11-363E7.4 was up-regulated for 32.49 times in SGC-7901 cells(p<0.05)and for 33.92 times in MGC-803 cells(p<0.05).3.Overexpression of lncRNA RP11-363E7.4 can inhibit proliferation and induce apoptosis of GC cells.Moreover,the protein expression level of Caspase-3,Bax and p53 were increased and that of Bcl-2 was decreased in the lncRNA RP11-363E7.4 overexpressed group compared to the negative control group.4.Overexpression of lncRNA RP11-363E7.4 can inhibit migration and invasion of GC cells.Moreover,the protein expression level of Ecadherin was increased and that of N-cadherin,Vimentin,Slug and β-catenin was decreased in the lncRNA RP11-363E7.4 overexpressed group compared to the negative control group.Conclusion:1.The expression level of lncRNA RP11-363E7.4 was significantly downregulated in gastric cancer tissues and cell lines than that of the corresponding normal ones,and the low expression level of RP11-363E7.4 was associated with T stage of tumor.2.LncRNA RP11-363E7.4 can inhibit proliferation,migration and invasion and induce apoptosis of gastric cancer cells to play a role of tumor suppression.3.LncRNA RP11-363E7.4 can influence the expression level of apoptosis related proteins and EMT related protein in gastric cancer cells,which may be related to its tumor suppression effect in gastric cancer cells.更多还原