NDRG3在胃癌中的作用及其机制的研究

发布时间：2019-05-28 18:00

【摘要】：目的:检测NDRG3在胃癌和癌旁组织、不同胃癌细胞株中的表达差异,探寻NDRG3在胃癌发生发展中的意义,为胃癌分子诊断与靶向治疗提供新的思路。方法:在数据库Oncomine中查询NDRG3在胃癌患者组织和对应癌旁组织中的mRNA水平的表达量差异,胃癌患者的总体生存率与NDRG3基因的表达量的关系。应用qRT-PCR和免疫组织化方法检测127例胃癌患者癌组织和对应癌旁组织中NDRG3在m RNA及蛋白水平的表达差异;Western blot验证比较胃癌细胞株中NDRG3表达异同。采用基因过表达技术构建NDRG3过表达质粒(pcDNA-NDRG3)并转染至SGC-7901细胞,用Western blot评价过表达的效果;CCK-8增殖实验和Transwell迁移实验观察其对胃癌细胞生长和迁移能力的影响;Western blot检测EMT和侵袭转移相关蛋白的变化,并在蛋白水平检测ERK1/2、PI3K/AKT通路蛋白的表达量的变化。运用RNA干扰(RNAi)技术抑制NDRG3在MGC-803胃癌细胞中的表达,并用Western blot验证抑制效果;平板克隆形成实验、CCK-8增殖实验、Transwell迁移、凋亡和细胞周期实验探讨NDRG3对胃癌细胞增殖、迁移能力的影响和胃癌细胞凋亡的变化;采用Western blot检测EMT、侵袭转移、凋亡等相关蛋白的表达及ERK1/2、PI3K/AKT通路的相关蛋白的表达,初步探讨NDRG3在胃癌组织中的作用机制。结果:1.qRT-PCR结果表明胃癌组织中NDRG3相对表达量高于对应的癌旁组织(P0.05),免疫组化结果发现NDRG3高表达于胃癌细胞(胞浆与胞核),而在癌旁组织中表达甚微,体外大部分胃癌细胞中的NDRG3的蛋白表达量高于正常胃上皮细胞GES-1中的表达量与数据库中统计的结果相一致。高表达的NDRG3的胃癌患者的总体生存率要低于低表达NDRG3的胃癌患者。2.Transwell迁移实验、CCK8增殖实验结果表明:过表达NDRG3的SGC-7901胃癌细胞迁移能力、增殖能力明显增强,使得E-cadherin表达降低、N-cadherin、β-catenin表达升高,促进EMT发生;Timp1表达降低、MMP9表达量升高,促进了肿瘤的侵袭转移。3.Transwell迁移实验、CCK8增殖实验、凋亡和周期实验结果表明:MGC-803细胞中NDRG3的表达被抑制后,抑制了细胞克隆、迁移、增殖能力,促进了胃癌细胞凋亡。E-cadherin表达上升,N-cadherin表达升高,抑制EMT发生;Timp1表达升高,抑制肿瘤的侵袭转移;PCNA表达降低,抑制肿瘤细胞的生长;BCL2L1表达降低,Cleaved-caspase 8、Cleaved caspase 3、Cleaved PARP表达增强,促进胃癌细胞凋亡。ERK1/2、PI3K/AKT相关通路中,p-ERK1/2、p-AKT、PI3K都显著降低。结论:NDRG3参与胃癌的发生、发展,可能通过激活ERK1/2、PI3K/AKT通路诱导胃癌细胞发生EMT促进肿瘤的转移和生长,抑制肿瘤细胞的凋亡。NDRG3将可作为一个新的胃癌分子诊断指标,进而为临床治疗胃癌提供新的依据。
[Abstract]:Objective: to detect the expression of NDRG3 in gastric cancer, paracancerous tissues and different gastric cancer cell lines, and to explore the significance of NDRG3 in the occurrence and development of gastric cancer, so as to provide a new idea for molecular diagnosis and targeted treatment of gastric cancer. Methods: the difference of mRNA level between gastric cancer patients and corresponding paracancerous tissues was investigated in database Oncomine, and the relationship between the overall survival rate of gastric cancer patients and the expression of NDRG3 gene was investigated. The expression of NDRG3 at m RNA and protein level in gastric cancer tissues and corresponding paracancerous tissues of 127 patients with gastric cancer was detected by qRT-PCR and immunohistology. the expression of NDRG3 in gastric cancer cell lines was compared by; Western blot. NDRG3 overexpression plasmid (pcDNA-NDRG3) was constructed by gene overexpression technique and transformed into SGC-7901 cells, and the effect of overexpression was evaluated by Western blot, and the effects of CCK-8 proliferation test and Transwell migration test on the growth and migration ability of gastric cancer cells were observed. The changes of EMT and invasion and metastasis related proteins were detected by Western blot, and the expression of ERK1/2,PI3K/AKT pathway proteins was detected at the protein level. RNA interference (RNAi) technique was used to inhibit the expression of NDRG3 in MGC-803 gastric cancer cells, and Western blot was used to verify the inhibitory effect. Plate clone formation assay, CCK-8 proliferation test, Transwell migration, apoptosis and cell cycle test were used to investigate the effects of NDRG3 on the proliferation and migration ability of gastric cancer cells and the changes of apoptosis of gastric cancer cells. Western blot was used to detect the expression of EMT, invasion and metastasis, apoptosis and ERK1/2,PI3K/AKT pathway related proteins, and to explore the mechanism of NDRG3 in gastric cancer. Results: the results of 1.qRT-PCR showed that the relative expression of NDRG3 in gastric cancer tissues was higher than that in corresponding paracancerous tissues (P 0.05). The results of immunohistochemistry showed that NDRG3 was highly expressed in gastric cancer cells (cytoplasm and nucleus), but very little in paracancerous tissues. The protein expression of NDRG3 in most gastric cancer cells in vitro was higher than that in normal gastric epithelial cells GES-1. The overall survival rate of gastric cancer patients with high expression of NDRG3 was lower than that of gastric cancer patients with low expression of NDRG3. 2. Transwell migration test, CCK8 proliferation test showed that the migration ability and proliferation ability of SGC-7901 gastric cancer cells overexpressing NDRG3 were significantly enhanced. The expression of E-cadherin was decreased, the expression of N-cadherin and 尾-catenin was increased, and the occurrence of EMT was promoted. The expression of Timp1 decreased and the expression of MMP9 increased, which promoted the invasion and metastasis of tumor. 3. Transwell migration test, CCK8 proliferation test, apoptosis and cycle test showed that the expression of NDRG3 in MGC-803 cells was inhibited, and the cell clone and migration were inhibited. The proliferation ability promoted the apoptosis of gastric cancer cells. The expression of E-cadherin increased, the expression of N-cadherin increased, and the occurrence of EMT was inhibited. The expression of Timp1 increased and the invasion and metastasis of tumor was inhibited, while the expression of PCNA decreased and the growth of tumor cells was inhibited. The expression of BCL2L1 decreased, the expression of Cleaved-caspase 8, cleaved caspase 3, and the expression of Cleaved-caspase PARP increased, which promoted the apoptosis of gastric cancer cells. ERK1 / 2, PI3K 鈮，

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