SIRT1在肺腺癌中的生物学作用及机制研究

发布时间：2018-06-15 13:51

本文选题：SIRT1 + 肺腺癌　；参考：《苏州大学》2016年博士论文

【摘要】：肺癌是全球范围内发病率及死亡率最高的肿瘤之一,严重危害人类健康。目前,外科手术、化疗、放疗仍是肺癌的主要治疗方式。近年,随着多学科综合治疗(multidisciplinary team,MDT)的发展,肺癌的治疗取得了长足的进步。然而,肺癌5年生存率仍徘徊在15%左右。精准医学模式是当今肿瘤治疗的趋势和方向,其关键是利用有效的生物标志物,区分目标人群,从而进行特异性个体化治疗。因此,寻找肺癌新的有效分子标志物以及潜在治疗靶点就显得尤为重要。随着人类基因组计划的完成,对肺癌在基因水平有了全新的认识。可以认为,在分子基因水平肺鳞癌和肺腺癌是“两种疾病”。现有资料也提示在肺腺癌中可能更易发现生物标志物,并可能从中获益。沉默信息调节因子1(silent information regulator 1,SIRT1)是一种功能类似烟酰胺腺嘌呤二核苷酸(NAD+)-依赖型组蛋白去乙酰化酶。SIRT1通过去乙酰化修饰作用,不仅作用于组蛋白,而且可作用于多种参与细胞衰老、凋亡、分化和肿瘤发生的基因及蛋白的表达。研究表明,SIRT1的表达水平与肿瘤密切相关,可能是肿瘤的潜在治疗靶点。有趣的是,SIRT1在不同类型的肿瘤中表现出截然不同的作用。一方面,SIRT1可能作为一种促癌因子下调p53等抑癌因子。另一方面,SIRT1也可抑制炎症和致癌的转录因子活性,以及通过维持基因稳定性发挥抑癌作用。近年,有少量研究报道探讨SIRT1在肺癌中作用,但结论并不一致。因此,有必要探讨SIRT1对肺腺癌生物学行为的影响及可能的分子机制,为阐明SIRT1在肺腺癌发生发展中的作用提供理论依据和实验基础。目的:1.观察SIRT1在肺腺癌组织中的表达情况,分析其表达水平与临床病理特征间的相关性,探讨其临床价值;2.研究SIRT1在肺腺癌中的生物学作用;3.探讨sirt1影响肺腺癌生物学行为的可能的分子机制。方法:1.应用免疫组织化学方法检测sirt1、survivin和vegf在肺腺癌组织芯片中肺癌组织、相应癌旁组织的表达情况,荧光原位杂交检测alk,egfr表达,统计分析sirt1与肺腺癌临床病理学特征间的相关性,揭示sirt1潜在的临床应用价值;2.应用cck-8法检测sirt1抑制剂(尼克酰胺、毒黄素)对肺腺癌a549细胞增殖的影响;3.apc-annexinv标记法,流式细胞仪检测sirt1抑制剂(尼克酰胺、毒黄素)对肺腺癌a549细胞凋亡的影响;4.划痕实验检测sirt1抑制剂(尼克酰胺、毒黄素)对肺腺癌a549细胞迁移的影响;5.利用人全基因组寡核苷酸微阵列芯片检测肺腺癌a549细胞及加入sirt1抑制剂(尼克酰胺、毒黄素)共培养的肺腺癌a549细胞mrna表达谱;6.利用生物信息学技术对差异表达基因进行通路分析和功能注释,筛选出在尼克酰胺组和毒黄素组均同向显著表达的差异基因作为sirt1的下游候选靶基因,并在tcga数据库验证sirt1与候选靶基因的相关性;7.利用rt-pcr和免疫印迹法检测加入尼克酰胺共培养的肺腺癌a549细胞中候选靶基因在mrna和蛋白水平的表达差异,初步探讨sirt1影响肺腺癌生物学行为的分子机制。结果:1.sirt1在肺腺癌中的表达及与临床病理特征间的相关性本研究中,sirt1在肺腺癌组织中表达显著高于相应癌旁正常组织。sirt1的表达与病理分期显著相关,但与性别,年龄,tnm分期,alk和egfr的表达状态无关。sirt1的表达与总生存期(os)相关,是肺腺癌预后不良的预测因子。研究发现sirt1与vegf的表达状态显著相关,但与survivin的表达状态无关。2.sirt1在肺腺癌中的生物学作用研究显示随着毒黄素浓度和作用时间的增加,肺腺癌a549细胞的增殖抑制率及凋亡率明显增加,并呈剂量及时间依赖关系。毒黄素组细胞迁移能力明显弱于对照组,差异有显著统计学意义。研究显示,尼克酰胺对肺腺癌A549细胞增殖的抑制率呈时间依赖关系,作用24小时时,尼克酰胺对肺腺癌A549细胞增殖的抑制率呈浓度依赖关系。随着尼克酰胺浓度和作用时间的增加,肺腺癌A549细胞凋亡率明显增加,并呈剂量及时间依赖关系。尼克酰胺组细胞迁移能力明显弱于对照组,差异有显著统计学意义。3.SIRT1影响肺腺癌生物学行为的可能的分子机制基因芯片mRNA表达谱检测发现,与肺腺癌A549细胞组比较毒黄素组共有745条差异基因表达上调倍值超过2倍,803条差异基因表达下调倍值超过2倍。与肺腺癌A549细胞组比较尼克酰胺组共有2505条差异基因表达上调倍值超过2倍,745条差异基因表达下调倍值超过2倍。分析同时在毒黄素组和尼克酰胺组显著同向表达的差异基因,发现在两组均表达上调的差异基因共159条,表达下调的差异基因共65条。由于SIRT1在肺腺癌中的生物学作用,进一步分析发现凋亡通路中的Bcl-2相关转录因子1(BCLAF1)可能是SIRT1下游靶基因。通过TCGA数据库分析发现SIRT1与BCLAF1均在肺腺癌组织共同表达,并且两者mRNA表达具有相关性。利用RT-PCR技术,发现加入尼克酰胺共培养后,肺腺癌A549细胞的BCLAF1基因mRNA表达水平显著升高。同时,免疫印迹试验显示BCLAF1蛋白水平也显著升高。结论:本研究证实SIRT1在肺腺癌组织中的表达明显高于相应的癌旁正常组织,并且是肺腺癌预后不良的预测因子。SIRT1抑制剂可以抑制肺腺癌A549细胞增殖、迁移,促进其凋亡。本研究利用人类全基因组寡核苷酸微阵列芯片,通过生物信息学技术筛选出差异基因:BCLAF1。并通过RT-PCR和免疫印迹法检测发现SIRT1抑制剂能显著升高肺腺癌A549细胞中BCLAF1基因mRNA和蛋白表达水平。因此,本研究提示SIRT1可能在肺腺癌的发生发展中扮演促癌基因的角色,其可能通过靶向调控BCLAF1参与肺腺癌的进展过程。这为进一步研究SIRT1的分子机制提供了理论基础和实验依据。
[Abstract]:Lung cancer is one of the highest incidence and mortality in the world, which seriously endangers human health. At present, surgery, chemotherapy and radiotherapy are still the main treatment methods for lung cancer. In recent years, with the development of multidisciplinary team (MDT), the treatment of lung cancer has made great progress. However, the 5 year survival rate of lung cancer The model of precision medicine is still around 15%. The model of precision medicine is the trend and direction of cancer treatment today. The key is to use effective biomarkers to distinguish the target population, so as to carry out specific individualized treatment. Therefore, it is particularly important to find new effective molecular markers and potential therapeutic targets for lung cancer. There is a new understanding of the genetic level of lung cancer. It is believed that at molecular level, lung squamous and adenocarcinoma are "two diseases". Existing data also suggest that biomarkers may be more likely to be found in lung adenocarcinoma and may benefit from it. Silencing information regulator 1 (silent information regulator 1, SIRT1) is a kind of disease. The function of nicotinamide adenine dinucleotide (NAD+) - dependent histone deacetylase.SIRT1 by deacetylation modification not only acts on histone, but also acts on the expression of genes and proteins involved in cell senescence, apoptosis, differentiation and tumor occurrence. The study shows that the expression level of SIRT1 is closely related to the tumor, It is possible to be a potential target for cancer treatment. Interestingly, SIRT1 plays a distinct role in different types of tumors. On the one hand, SIRT1 may downregulate p53 as a cancer factor. On the other hand, SIRT1 can also inhibit the activity of the transcription factor of inflammatory and carcinogenic, and the inhibition of cancer by maintaining the stability of the gene. In recent years, a small number of studies have been reported to explore the role of SIRT1 in lung cancer, but the conclusions are not consistent. Therefore, it is necessary to explore the effect of SIRT1 on the biological behavior of lung adenocarcinoma and the possible molecular mechanism to provide a theoretical basis and a practical basis for elucidating the role of SIRT1 in the development of lung adenocarcinoma. Objective: 1. to observe SIRT1 in the lung adenocarcinoma tissue. The correlation between the expression level and the clinicopathological features was analyzed and its clinical value was discussed. 2. the biological role of SIRT1 in lung adenocarcinoma was studied. 3. the possible molecular mechanism of SIRT1 to affect the biological behavior of lung adenocarcinoma. Method: 1. to detect SIRT1, survivin and VEGF in the tissue core of lung adenocarcinoma with immunohistochemical method. The expression of lung cancer tissue and corresponding paracancerous tissue, fluorescence in situ hybridization detection ALK, EGFR expression, statistical analysis of the correlation between SIRT1 and the clinicopathological features of lung adenocarcinoma, to reveal the potential clinical value of SIRT1; 2. the effect of SIRT1 inhibitor (Nick amide, TOXOFLAVIN) on the proliferation of lung adenocarcinoma A549 cells by CCK-8; 3.ap C-annexinv labeling method, flow cytometry to detect the effect of SIRT1 inhibitor (Nik amide, TOXOFLAVIN) on the apoptosis of lung adenocarcinoma A549 cells; 4. scratch test to detect the effect of SIRT1 inhibitor (Nik amide, TOXOFLAVIN) on the migration of A549 cells in lung adenocarcinoma cells; 5. the detection of lung adenocarcinoma A549 cells and Si by human genome oligonucleotide microarray RT1 inhibitor (niacin, poison Huang Su) co cultured mRNA expression profiles of lung adenocarcinoma A549 cells; 6. using bioinformatics technology to carry out pathway analysis and functional annotation of differentially expressed genes, screening the differentially expressed differentially expressed genes in the niacin group and the poison Huang Su group as the downstream candidate target genes of SIRT1, and in the TCGA database To verify the correlation between SIRT1 and candidate target genes; 7. the difference in the expression of candidate target genes in the mRNA and protein levels of lung adenocarcinoma A549 cells co cultured with RT-PCR and Western blot was used to investigate the molecular mechanism of SIRT1 on the biological behavior of lung adenocarcinoma. The expression and clinical significance of 1.sirt1 in lung adenocarcinoma In the correlation between pathological features, the expression of SIRT1 in lung adenocarcinoma was significantly higher than that of.Sirt1 in the normal tissue adjacent to the corresponding cancerous tissue. The expression of.Sirt1 was related to the state of sex, age, TNM staging, ALK and EGFR expression and the total survival time (OS), which was a predictor of poor prognosis in lung adenocarcinoma. It was found that the expression of SIRT1 was significantly related to the expression state of VEGF, but it was not related to the expression state of Survivin in the biological action of.2.sirt1 in lung adenocarcinoma. The proliferation inhibition rate and apoptosis rate of A549 cells in lung adenocarcinoma cells increased significantly with the increase of the concentration of TOXOFLAVIN and the time of action, and there was a dose and time dependence. The cell migration of the TOXOFLAVIN group The effect of niacin on the proliferation of lung adenocarcinoma A549 cells was time dependent. The inhibitory rate of niacin on the proliferation of lung adenocarcinoma A549 cells was in a concentration dependent relationship at 24 hours. With the increase of nicotamide concentration and time, the lung adenocarcinoma was increased. The apoptosis rate of A549 cells increased significantly and was dependent on the dose and time dependence. The cell migration ability of niacin group was significantly weaker than that of the control group. The difference has significant statistical significance in the possible molecular mechanism gene chip mRNA expression detection of.3.SIRT1, which affects the biological behavior of lung adenocarcinoma, and compared with the A549 cell group of lung adenocarcinoma. The up regulation of 745 differentially expressed genes was more than 2 times, and the downregulation of 803 differentially expressed genes exceeded 2 times. Compared with the A549 cell group of lung adenocarcinoma, there were 2505 different genes expression up-regulation times more than 2 times and 745 differentially expressed downregulation times more than 2 times. A total of 159 differentially expressed genes were expressed in all two groups and 65 differentially expressed genes were expressed in the two groups. The Bcl-2 related transcriptional factor 1 (BCLAF1) in the apoptotic pathway may be the target gene of the downstream SIRT1 because of the biological function of the expression in the lung adenocarcinoma. The TCGA database analysis found SIRT1. The expression of mRNA expression was associated with BCLAF1 in lung adenocarcinoma tissue and the expression of both mRNA was correlated. The BCLAF1 gene expression level of A549 cells in lung adenocarcinoma was significantly increased after co culture with nicotinamide. Meanwhile, the Western blot test showed that the level of BCLAF1 protein was also significantly elevated. Conclusion: This study confirmed that SIRT1 is in the lung. The expression in adenocarcinoma tissue is significantly higher than that of the corresponding normal tissue adjacent to the carcinoma, and the predictive factor.SIRT1 inhibitor for the poor prognosis of lung adenocarcinoma can inhibit the proliferation, migration and apoptosis of lung adenocarcinoma A549 cells. This study uses the whole human genome oligonucleotide microarray to screen out the differential genes through bioinformatics Technology: BCLA F1. and RT-PCR and immunoblotting detected that SIRT1 inhibitors can significantly increase the level of mRNA and protein expression of BCLAF1 gene in A549 cells of lung adenocarcinoma. Therefore, this study suggests that SIRT1 may play a role in the development of lung adenocarcinoma in the development of lung adenocarcinoma, which may be involved in the progression of lung adenocarcinoma by targeting BCLAF1. Further studies on the molecular mechanism of SIRT1 provide a theoretical basis and experimental evidence.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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