【作者】 张琳；

【导师】 张桂珍；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2018， 博士

【摘要】 背景:肺癌是世界范围内发病率和死亡率均较高的恶性疾病之一,而非小细胞肺癌(Non-small-cell lung cancer,NSCLC)是其最常见的类型,约占所有肺癌病例的80%。肺癌常规的治疗方法包括外科治疗、放射治疗、化学治疗,随着分子生物学的迅猛发展,靶向治疗正在逐步成为肺癌的主要治疗手段之一。因此,寻找NSCLC发病过程中的关键基因和相关信号通路,并揭示其中的分子机制,对于NSCLC靶向治疗至关重要。研究者们先已发现了许多在NSCLC发生、发展过程中起决定作用的癌基因和癌相关蛋白,为治疗提供了新的研究和治疗思路。水通道蛋白5(Aquaporin 5,AQP5)一种跨细胞膜的水通道蛋白,具有调节细胞内水平衡的作用。近期有研究表明,AQP5在多种肿瘤细胞中表达上调,可促进恶性肿瘤细胞的增殖,对其进行基因沉默可抑制肿瘤生长。但AQP5与NSCLC之间的相关性仍有待探索。目的:在国内外研究的基础上,通过基因工程重组技术沉默AQP5基因在NSCLC细胞中的表达,系统评价AQP5对NSCLC细胞增殖凋亡、迁徙侵袭等生物学功能的影响,并进一步探讨AQP5基因沉默后对相关功能蛋白的及信号转导通路的影响,阐述AQP5与NSCLC发生发展的密切关系及相关的信号转导通路及分子机制,为NSCLC的治疗提供新靶点。方法:1.Western Blot方法检测正常人肺细胞MRC-5和人肺癌细胞系HCC827、A549、H1299、H358中AQP5的表达水平,筛选出AQP5高表达细胞系A549。设计并构建特异性真核表达质粒AQP5-p GCH1/Neo和阴性质粒NC-p GCH1/Neo,利用脂质体转染入A549细胞,构建AQP5基因沉默细胞及阴性对照组细胞。Real-time PCR和Western Blot方法检测并比较未处理细胞组、阴性对照组及基因沉默组细胞的AQP5基因和蛋白表达水平。2.利用细胞集落形成实验,MTT法检测各组细胞增殖活力,采用流式细胞术考察细胞周期分布及细胞凋亡差异;Western Blot法检测细胞凋亡通路相关蛋白Bcl-2、Cleaved caspased-3和Bax的表达情况。3.划痕实验观察三组细胞的迁移能力,Transwell侵袭实验观察侵袭能力,Western Blot法检测肿瘤转移相关蛋白MMP-2,MMP-9和Vimentin表达水平。4.利用Western Blot对ERK1/2信号通路相关蛋白p-ERK、p-CREB和c-fos表达量进行检测,探讨AQP5对ERK1/2信号通路的影响。5.在上述体外实验的基础上,将未处理细胞组、阴性对照组及基因沉默组三组细胞接种于裸鼠皮下,建立体内异种移植瘤模型,观察体内情况下AQP5表达下调对肿瘤细胞的增殖和凋亡影响。结果:1.成功构建含有AQP5 sh RNA的AQP5-p GCH1/Neo质粒和阴性对照质粒NC-p GCH1/Neo,并转染至A549细胞;转染AQP5-p GCH1/Neo质粒的A549细胞中AQP5基因和蛋白表达水平均显著降低。2.细胞集落实验表明,AQP5 sh RNA组形成细胞集落能力显著下降;MTT实验表明AQP5 sh RNA细胞组各时间点吸光度均降低,时间越长,差异越显著。流式细胞仪结果表明AQP5 sh RNA组细胞阻滞在G0/G1期,而在S期、G2/M期细胞数量显著减低,并且凋亡显著。凋亡相关蛋白检测结果显示AQP5 sh RNA组Bcl-2表达下调,Cleaved caspased-3和Bax表达上调,Bcl-2/Bax比值降低,说明AQP5基因沉默抑制肿瘤细胞增殖,阻滞细胞周期,抑制肿瘤细胞分裂,并可通过调控Bcl-2家族凋亡相关蛋白促进A549细胞凋亡。3.细胞迁移实验结果显示AQP5 sh RNA组细胞迁移能力下降。Transwell细胞侵袭实验表明AQP5 sh RNA组进入下层小室细胞减少,侵袭能力下降。Western Blot结果显示肿瘤转移关键因子MMP-2和MMP-9和间质细胞转化标志蛋白Vimentin表达降低,说明AQP5表达下调降低肿瘤细胞侵袭能力,抑制肿瘤细转移。4.AQP5 sh RNA组细胞p-ERK、p-CREB和c-fos表达量均显著降低。说明AQP5表达下调可降低ERK1/2信号通路蛋白磷酸化,抑制ERK1/2信号通路。5.AQP5 sh RNA组小鼠的肿瘤体积、重量显著小于对照组,肿瘤生长速度明显下降;Tunel法检测肿瘤凋亡结果显示AQP5 sh RNA组小鼠肿瘤组织中凋亡细胞更多。说明在体内环境下,AQP5基因沉默可抑制肿瘤增殖,促进肿瘤凋亡。结论:1.使用RNAi技术建立了非小细胞肺癌A549细胞的AQP5基因沉默细胞系。2.证实AQP5与非小细胞肺癌A549生物学功能关系密切,表达下调抑制肿瘤细胞的增殖、迁移、侵袭,促进肿瘤细胞凋亡,并能调控相关功能蛋白的表达水平,对ERK1/2信号通路有抑制作用。3.为非小细胞肺癌的诊断和治疗提供新的潜在标志物及靶点。4.为RNA干扰技术治疗肿瘤疾病提供实验研究数据。更多还原

【Abstract】 Background:Lung cancer is one of the most common malignancies with high morbidity and mortality worldwide.Non-small-cell lung cancer(NSCLC)is the most common type,accounting for approximately 80% of all lung cancer cases..Conventional treatment methods for lung cancer include surgical treatment,radiation therapy,and chemotherapy.With the rapid development of molecular biology,targeted gene therapies are gradually becoming one of the main treatment methods for lung cancer,and certain effects have achieved.Researchers have discovered many oncogenes and cancer-associated proteins that play a decisive role in the development and progression of NSCLC,providing new research and treatment ideas for the treatment of NSCLC.Therefore,the search for key genes and related signaling pathways in the pathogenesis of NSCLC,and reveal the molecular mechanisms,are essential for targeted therapy.Aquaporin 5(AQP5)is an aquaporin transmembrane that regulates intracellular water balance.Recent studies have shown that AQP5 is upregulated in a variety of tumor cells and can promote the proliferation of malignant tumor cells,and gene silencing can inhibit tumor growth.However,the correlation between AQP5 and NSCLC remains to be explored.Purpose:Based on the research at home and abroad,the expression of AQP5 gene in non-small cell lung cancer cells was silenced by genetic engineering recombination technology,and the effects of AQP5 on tumor cell proliferation,apoptosis,migration,and invasion were explored.Further study on the effect of AQP5 gene silencing on related functional proteins and ERK1/2 signal transduction pathways,and explore the role of AQP5 and NSCLC in the development and the involved signal pathways and molecular mechanisms,provide a new direction for the targeted treatment of NSCLC.Method:Western Blot analysis was used to detect the expression of AQP5 in normal human lung cells MRC-5 and human lung cancer cell lines HCC827,A549,H1299,and H358.AQP5 high expression cell line A549 was selected.The specific eukaryotic expression plasmids AQP5-p GCH1/Neo(sh AQP5)and NC-p GCH1/Neo(sh NC)were designed and constructed.The liposomes were transfected into A549 cells and stable transfected cell lines were obtained by screening.Real-time PCR and Western Blot were used to detect the gene and protein expression levels of AQP5 in the untreated cell group,blank control group,and gene silencing group.2.Use cell colony formation assays to test the proliferation of the three groups of cells.MTT assay was used to detect the cell proliferation activity.Flow cytometry was used to investigate the cell cycle distribution and apoptosis.The expression of apoptosis-related proteins Bcl-2,Cleaved caspased-3 and Bax was detected by Western Blot.3.Cell wound scratch assay to observe the migration ability of the three groups of cells,Transwell invasion experiment to observe the invasive ability of each group of cells,Western Blot assay to detect cell migration and invasion-related proteins,MMP-2,MMP-9 and Vimentin expression levels.Western Blot was used to detect the expression of p-ERK,p-CREB and c-fos related proteins in ERK1/2 signaling pathway.Explore the signaling pathways that AQP5 may affect.On the basis of the above in vitro experiments,three kinds of cells were inoculated subcutaneously in nude mice to establish a xenotransplantation model in vivo,and the tumor growth curve was observed and recorded.Tumor tissue was removed 30 days after inoculation and weighed to compare proliferative capacity.Three sections of tumor tissue were taken for Tunel to detect the apoptosis of tumor cells.Result:1.AQP5-p GCH1/Neo plasmid containing AQP5 sh RNA and control plasmid NC-p GCH1/Neo were successfully constructed and stably transfected into A549 cells;AQP5 m RNA and protein expression levels were significantly reduced in A549 cells after transfection.2.Cell colony experiments showed that colony formation ability of AQP5 sh RNA cells decreased significantly;MTT assay showed that the absorbance of AQP5 sh RNA cells decreased at each time point,and the longer the time,the more significant the difference.The cell cycle was detected by flow cytometry.The results showed that AQP5 sh RNA cells were arrested in G0/G1 phase,while the number of cells in S phase and G2/M phase was significantly decreased.Flow cytometry revealed significant apoptosis in AQP5 sh RNA cells.Apoptosis-related protein assays showed that Bcl-2 expression was down-regulated and Cleaved caspased-3 and Bax were up-regulated,indicating that AQP5 silencing inhibited tumor cell proliferation,inhibited tumor cell division,and promoted apoptosis of A549 cells by affecting apoptosis-related protein expression..3.Cell migration experiments showed that the migration ability of AQP5 sh RNA cells was decreased.Transwell cell invasion assays showed that the AQP5 sh RNA group entered the lower chamber cells and the invasive ability decreased.Western Blot results showed that the expression of MMP-2 and MMP-9 as key factors of migration and Vimentin expression of mesenchymal transition protein decreased,indicating that AQP5 protein down-regulates tumor cell migration and invasion,and inhibits epithelial-mesenchymal process of tumor cells.4.The expression of p-ERK,p-CREB and c-fos in AQP5 sh RNA group was significantly reduced.This indicates that the down-regulation of AQP5 can reduce the phosphorylation of ERK1/2 signaling pathway and inhibit the ERK1/2 signaling pathway.5.The tumor volume and weight of mice in AQP5 sh RNA group were significantly smaller than those in the control group,and the tumor growth rate was significantly decreased.Tunel assay detected tumor apoptosis results showed that there were more apoptotic cells in tumor tissue of AQP5 sh RNA group mice.It shows that AQP5 gene silencing can inhibit tumor proliferation and promote tumor apoptosis in vivo.Conclusion:1.AQP5 gene silencing cell line of non-small cell lung cancer A549 cells was established using RNA interference technology.2.It was confirmed that AQP5 is closely related to the biological function of non-small cell lung cancer A549.The down-regulated expression of AQP5 inhibits the proliferation,migration and invasion of tumor cells,promotes the apoptosis of tumor cells,and regulates the expression level of related functional proteins,and inhibits the ERK1/2 signaling pathway.3.Provide new potential markers and targets for the diagnosis and treatment of non-small cell lung cancer.4.Provide experimental data for RNA interference treatment of tumor diseases.更多还原