【作者】 吴越；

【导师】 邵世和；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2021， 硕士

【摘要】 目的:明确DRAM1在胃癌及相应癌旁组织、正常胃黏膜上皮细胞及胃癌细胞系中的表达差异,探究DRAM1蛋白在胃癌细胞增殖迁移过程中的作用及可能的作用机制,希望能为胃癌的诊断和治疗提供新的思路和靶点。方法:(1)通过在线数据库分析DRAM1在胃癌和正常胃组织中的m RNA水平,应用qRT-PCR检测临床胃癌组织及相应的癌旁组织中DRAM1的m RNA水平,并用Western blotting技术检测蛋白水平差异。(2)运用qRT-PCR和Western blotting对正常胃上皮细胞及各胃癌细胞株中DRAM1表达进行差异分析。筛选DRAM1高表达的胃癌细胞SGC-7901转染小干扰RNA(si DRAM1),对表达量相对较低的胃癌细胞HGC-27转染过表达质粒(pcDNA3.1-DRAM1),构建DRAM1体外敲减及过表达细胞模型并验证其效率。(3)通过CCK-8及细胞集落形成实验检测干扰或上调DRAM1表达对胃癌细胞的增殖能力的影响;细胞划痕愈合及Transwell迁移实验观察DRAM1对胃癌细胞迁移的影响;蛋白免疫印迹实验检测上皮间充质转化(EMT)、细胞增殖、迁移相关蛋白以及Wnt/β-catenin通路蛋白,探究DRAM1在胃癌细胞增殖和迁移中的调控作用。结果:(1)荧光定量PCR与Western blotting结果显示:DRAM1在胃癌组织中的表达量普遍高于相应癌旁组织。细胞系蛋白印迹实验结果显示,DRAM1在胃癌细胞中的表达上调,尤其在SGC-7901细胞中表达最高,在HGC-27细胞中表达相对较低。(2)CCK-8增殖实验与细胞克隆形成实验结果显示:降低SGC-7901细胞中DRAM1水平后细胞增殖减弱。通过细胞划痕实验及Transwell迁移实验观察到:敲减DRAM1抑制了SGC-7901细胞的运动能力,细胞迁移显著减弱。Western blotting实验显示:下调DRAM1水平后,SGC-7901细胞中上皮细胞标志性蛋白E-cadherin表达上调,同时伴有间叶细胞标志蛋白Vimentin、N-cadherin蛋白水平降低,说明DRAM1下调抑制了EMT;基质金属蛋白酶MMP2、MMP9及增殖细胞核抗原PCNA显著减少,表明胃癌细胞增殖及迁移受到抑制;通路蛋白GSK3β的变化无统计学意义,β-catenin、Cyclin D1、p-GSK3β、c-Myc表达减弱,表明Wnt/β-catenin信号通路受到抑制。(3)过表达质粒pcDNA3.1-DRAM1转染HGC-27细胞后,细胞功能实验显示胃癌细胞增殖、迁移能力显著增强。Western blotting结果显示:E-cadherin表达减少,Vimentin、N-cadherin水平升高,EMT受到促进;细胞增殖、迁移相关蛋白表达上升;Wnt/β-catenin信号通路关键蛋白水平升高,表明信号通路受到激活。结论:相较于相应癌旁组织或正常胃上皮细胞,DRAM1在胃癌组织及胃癌细胞系中表达上调。在胃癌细胞中过表达DRAM1可增强细胞的增殖、迁移能力,促进细胞发生EMT;敲减DRAM1抑制胃癌细胞增殖迁移,抑制EMT发生。此外,Wnt/β-catenin信号通路可能为DRAM1促进胃癌的发生发展途径。更多还原

【Abstract】 Objective:To detect the differential expression of DRAM1 in gastric tissues and tissues adjacent to cancer,and between normal gastric epithelial cells and gastric cancer cell lines.To understand the impact of DRAM1 on migration and proliferation of gastric cancer cells and the potential mechanism.To hopefully offer a fresh idea and target for future diagnosis and treatment of gastric cancer.Methods:(1)Online public databases were used to evaluate the expression level of DRAM1 in gastric cancer and normal gastric tissues.Both the m RNA and protein levels of DRAM1 were detected respectively using quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting,in order to compare the differential expression between gastric cancer tissues and adjacent nontumorous tissues.(2)The DRAM1 levels in normal gastric epithelial cells and in gastric cancer cell lines were assessed by means of qRT-PCR and Western blotting.The small interfering RNA(si DRAM1)was transfected into SGC-7901 cells which showed high expression of DRAM1,while the plasmid(pcDNA3.1-DRAM1)was transfected into HGC-27 cells where DRAM1 expression was relatively low,so that the model cells with low/high DRAM1 expression were built.The efficiency of knockdown or overexpression was examined by Western blotting.(3)The effect of DRAM1 on the proliferative potential of gastric cancer cells was evaluated by Cell Counting Kit-8 assay and cell clone formation assay,after DRAM1 knockdown or overexpression.Wound healing assay together with Transwell assay were performed to observe the changes in migration of SGC-7901 and HGC-27 cells after DRAM1 knockdown/overexpression.Western blotting assay was used to detect EMT(Epithelial-Mesenchymal Transition),cell proliferation and migration related proteins,and Wnt/β-catenin pathway proteins to explore the regulatory role of DRAM1 in the proliferation and migration of gastric cancer cells.Results:(1)qRT-PCR and Western blotting results indicated that in comparison to the adjacent tissues,the expression of DRAM1 was generally increased in gastric cancer tissues.Western blotting experiments found that the protein level of DRAM1 in gastric carcinoma cells was significantly higher than that in normal gastric epithelial cells,with the highest expression in SGC-7901 cells while a relatively low expression in HGC-27 cells.(2)The consistent results of CCK-8 assay and cell clone formation assay revealed the decrease in proliferation of SGC-7901 cells after transfected with si-DRAM1.The migration of SGC-7901 cells was also significantly inhibited after DRAM1 knockdown,as shown by the results of wound healing and Transwell migration assay.In addition,Western blotting results showed that the expression of E-cadherin,an epithelial marker,in SGC-7901 cells was upregulated due to knockdown of DRAM1,while the levels of Vimentin and N-cadherin,both are interstitial markers,were decreased,indicating that the downregulation of DRAM1 inhibited EMT.Matrix metalloproteinases MMP2,MMP9 and PCNA(proliferating nuclear antigen)were significantly decreased,indicating that the proliferation and migration of cells were restrained.The expression of β-catenin,Cyclin D1,p-GSK3βand C-Myc were downregulated,with no significant variation in GSK3β,showing that the Wnt/β-catenin signaling pathway was inhibited.(3)The cell function experiments revealed that,after transfection of HGC-27 cells with the overexpressed plasmid pcDNA3.1-DRAM1,the proliferation and migration ability of gastric cancer cells were significantly enhanced.Western blotting results consistently showed that EMT,cell proliferation and migration,and Wnt/β-catenin signaling pathway protein levels were changed accordingly.Conclusion:DRAM1 was up-regulated in gastric cancer tissues and cells in comparison with para-carcinoma tissues and normal gastric epithelial cells.In vitro gastric cancer cells,DRAM1 overexpression strengthened the cell proliferation as well as migration,and facilitated EMT.Knockdown of DRAM1 inhibited proliferation,migration,and EMT of gastric cancer cells.Mechanistically,DRAM1 may promote the occurrence and progression of gastric cancer via Wnt/β-catenin signaling pathway.更多还原