【摘要】：目的:檢測(cè)NDRG3在胃癌和癌旁組織、不同胃癌細(xì)胞株中的表達(dá)差異,探尋NDRG3在胃癌發(fā)生發(fā)展中的意義,為胃癌分子診斷與靶向治療提供新的思路。方法:在數(shù)據(jù)庫Oncomine中查詢NDRG3在胃癌患者組織和對(duì)應(yīng)癌旁組織中的mRNA水平的表達(dá)量差異,胃癌患者的總體生存率與NDRG3基因的表達(dá)量的關(guān)系。應(yīng)用qRT-PCR和免疫組織化方法檢測(cè)127例胃癌患者癌組織和對(duì)應(yīng)癌旁組織中NDRG3在m RNA及蛋白水平的表達(dá)差異;Western blot驗(yàn)證比較胃癌細(xì)胞株中NDRG3表達(dá)異同。采用基因過表達(dá)技術(shù)構(gòu)建NDRG3過表達(dá)質(zhì)粒(pcDNA-NDRG3)并轉(zhuǎn)染至SGC-7901細(xì)胞,用Western blot評(píng)價(jià)過表達(dá)的效果;CCK-8增殖實(shí)驗(yàn)和Transwell遷移實(shí)驗(yàn)觀察其對(duì)胃癌細(xì)胞生長和遷移能力的影響;Western blot檢測(cè)EMT和侵襲轉(zhuǎn)移相關(guān)蛋白的變化,并在蛋白水平檢測(cè)ERK1/2、PI3K/AKT通路蛋白的表達(dá)量的變化。運(yùn)用RNA干擾(RNAi)技術(shù)抑制NDRG3在MGC-803胃癌細(xì)胞中的表達(dá),并用Western blot驗(yàn)證抑制效果;平板克隆形成實(shí)驗(yàn)、CCK-8增殖實(shí)驗(yàn)、Transwell遷移、凋亡和細(xì)胞周期實(shí)驗(yàn)探討NDRG3對(duì)胃癌細(xì)胞增殖、遷移能力的影響和胃癌細(xì)胞凋亡的變化;采用Western blot檢測(cè)EMT、侵襲轉(zhuǎn)移、凋亡等相關(guān)蛋白的表達(dá)及ERK1/2、PI3K/AKT通路的相關(guān)蛋白的表達(dá),初步探討NDRG3在胃癌組織中的作用機(jī)制。結(jié)果:1.qRT-PCR結(jié)果表明胃癌組織中NDRG3相對(duì)表達(dá)量高于對(duì)應(yīng)的癌旁組織(P0.05),免疫組化結(jié)果發(fā)現(xiàn)NDRG3高表達(dá)于胃癌細(xì)胞(胞漿與胞核),而在癌旁組織中表達(dá)甚微,體外大部分胃癌細(xì)胞中的NDRG3的蛋白表達(dá)量高于正常胃上皮細(xì)胞GES-1中的表達(dá)量與數(shù)據(jù)庫中統(tǒng)計(jì)的結(jié)果相一致。高表達(dá)的NDRG3的胃癌患者的總體生存率要低于低表達(dá)NDRG3的胃癌患者。2.Transwell遷移實(shí)驗(yàn)、CCK8增殖實(shí)驗(yàn)結(jié)果表明:過表達(dá)NDRG3的SGC-7901胃癌細(xì)胞遷移能力、增殖能力明顯增強(qiáng),使得E-cadherin表達(dá)降低、N-cadherin、β-catenin表達(dá)升高,促進(jìn)EMT發(fā)生;Timp1表達(dá)降低、MMP9表達(dá)量升高,促進(jìn)了腫瘤的侵襲轉(zhuǎn)移。3.Transwell遷移實(shí)驗(yàn)、CCK8增殖實(shí)驗(yàn)、凋亡和周期實(shí)驗(yàn)結(jié)果表明:MGC-803細(xì)胞中NDRG3的表達(dá)被抑制后,抑制了細(xì)胞克隆、遷移、增殖能力,促進(jìn)了胃癌細(xì)胞凋亡。E-cadherin表達(dá)上升,N-cadherin表達(dá)升高,抑制EMT發(fā)生;Timp1表達(dá)升高,抑制腫瘤的侵襲轉(zhuǎn)移;PCNA表達(dá)降低,抑制腫瘤細(xì)胞的生長;BCL2L1表達(dá)降低,Cleaved-caspase 8、Cleaved caspase 3、Cleaved PARP表達(dá)增強(qiáng),促進(jìn)胃癌細(xì)胞凋亡。ERK1/2、PI3K/AKT相關(guān)通路中,p-ERK1/2、p-AKT、PI3K都顯著降低。結(jié)論:NDRG3參與胃癌的發(fā)生、發(fā)展,可能通過激活ERK1/2、PI3K/AKT通路誘導(dǎo)胃癌細(xì)胞發(fā)生EMT促進(jìn)腫瘤的轉(zhuǎn)移和生長,抑制腫瘤細(xì)胞的凋亡。NDRG3將可作為一個(gè)新的胃癌分子診斷指標(biāo),進(jìn)而為臨床治療胃癌提供新的依據(jù)。
[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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