本文選題：SIRT1 + 肺腺癌　； 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：肺癌是全球范圍內(nèi)發(fā)病率及死亡率最高的腫瘤之一,嚴(yán)重危害人類健康。目前,外科手術(shù)、化療、放療仍是肺癌的主要治療方式。近年,隨著多學(xué)科綜合治療(multidisciplinary team,MDT)的發(fā)展,肺癌的治療取得了長(zhǎng)足的進(jìn)步。然而,肺癌5年生存率仍徘徊在15%左右。精準(zhǔn)醫(yī)學(xué)模式是當(dāng)今腫瘤治療的趨勢(shì)和方向,其關(guān)鍵是利用有效的生物標(biāo)志物,區(qū)分目標(biāo)人群,從而進(jìn)行特異性個(gè)體化治療。因此,尋找肺癌新的有效分子標(biāo)志物以及潛在治療靶點(diǎn)就顯得尤為重要。隨著人類基因組計(jì)劃的完成,對(duì)肺癌在基因水平有了全新的認(rèn)識(shí)�？梢哉J(rèn)為,在分子基因水平肺鱗癌和肺腺癌是“兩種疾病”�，F(xiàn)有資料也提示在肺腺癌中可能更易發(fā)現(xiàn)生物標(biāo)志物,并可能從中獲益。沉默信息調(diào)節(jié)因子1(silent information regulator 1,SIRT1)是一種功能類似煙酰胺腺嘌呤二核苷酸(NAD+)-依賴型組蛋白去乙�；�。SIRT1通過(guò)去乙�；揎椬饔�,不僅作用于組蛋白,而且可作用于多種參與細(xì)胞衰老、凋亡、分化和腫瘤發(fā)生的基因及蛋白的表達(dá)。研究表明,SIRT1的表達(dá)水平與腫瘤密切相關(guān),可能是腫瘤的潛在治療靶點(diǎn)。有趣的是,SIRT1在不同類型的腫瘤中表現(xiàn)出截然不同的作用。一方面,SIRT1可能作為一種促癌因子下調(diào)p53等抑癌因子。另一方面,SIRT1也可抑制炎癥和致癌的轉(zhuǎn)錄因子活性,以及通過(guò)維持基因穩(wěn)定性發(fā)揮抑癌作用。近年,有少量研究報(bào)道探討SIRT1在肺癌中作用,但結(jié)論并不一致。因此,有必要探討SIRT1對(duì)肺腺癌生物學(xué)行為的影響及可能的分子機(jī)制,為闡明SIRT1在肺腺癌發(fā)生發(fā)展中的作用提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。目的:1.觀察SIRT1在肺腺癌組織中的表達(dá)情況,分析其表達(dá)水平與臨床病理特征間的相關(guān)性,探討其臨床價(jià)值;2.研究SIRT1在肺腺癌中的生物學(xué)作用;3.探討sirt1影響肺腺癌生物學(xué)行為的可能的分子機(jī)制。方法:1.應(yīng)用免疫組織化學(xué)方法檢測(cè)sirt1、survivin和vegf在肺腺癌組織芯片中肺癌組織、相應(yīng)癌旁組織的表達(dá)情況,熒光原位雜交檢測(cè)alk,egfr表達(dá),統(tǒng)計(jì)分析sirt1與肺腺癌臨床病理學(xué)特征間的相關(guān)性,揭示sirt1潛在的臨床應(yīng)用價(jià)值;2.應(yīng)用cck-8法檢測(cè)sirt1抑制劑(尼克酰胺、毒黃素)對(duì)肺腺癌a549細(xì)胞增殖的影響;3.apc-annexinv標(biāo)記法,流式細(xì)胞儀檢測(cè)sirt1抑制劑(尼克酰胺、毒黃素)對(duì)肺腺癌a549細(xì)胞凋亡的影響;4.劃痕實(shí)驗(yàn)檢測(cè)sirt1抑制劑(尼克酰胺、毒黃素)對(duì)肺腺癌a549細(xì)胞遷移的影響;5.利用人全基因組寡核苷酸微陣列芯片檢測(cè)肺腺癌a549細(xì)胞及加入sirt1抑制劑(尼克酰胺、毒黃素)共培養(yǎng)的肺腺癌a549細(xì)胞mrna表達(dá)譜;6.利用生物信息學(xué)技術(shù)對(duì)差異表達(dá)基因進(jìn)行通路分析和功能注釋,篩選出在尼克酰胺組和毒黃素組均同向顯著表達(dá)的差異基因作為sirt1的下游候選靶基因,并在tcga數(shù)據(jù)庫(kù)驗(yàn)證sirt1與候選靶基因的相關(guān)性;7.利用rt-pcr和免疫印跡法檢測(cè)加入尼克酰胺共培養(yǎng)的肺腺癌a549細(xì)胞中候選靶基因在mrna和蛋白水平的表達(dá)差異,初步探討sirt1影響肺腺癌生物學(xué)行為的分子機(jī)制。結(jié)果:1.sirt1在肺腺癌中的表達(dá)及與臨床病理特征間的相關(guān)性本研究中,sirt1在肺腺癌組織中表達(dá)顯著高于相應(yīng)癌旁正常組織。sirt1的表達(dá)與病理分期顯著相關(guān),但與性別,年齡,tnm分期,alk和egfr的表達(dá)狀態(tài)無(wú)關(guān)。sirt1的表達(dá)與總生存期(os)相關(guān),是肺腺癌預(yù)后不良的預(yù)測(cè)因子。研究發(fā)現(xiàn)sirt1與vegf的表達(dá)狀態(tài)顯著相關(guān),但與survivin的表達(dá)狀態(tài)無(wú)關(guān)。2.sirt1在肺腺癌中的生物學(xué)作用研究顯示隨著毒黃素濃度和作用時(shí)間的增加,肺腺癌a549細(xì)胞的增殖抑制率及凋亡率明顯增加,并呈劑量及時(shí)間依賴關(guān)系。毒黃素組細(xì)胞遷移能力明顯弱于對(duì)照組,差異有顯著統(tǒng)計(jì)學(xué)意義。研究顯示,尼克酰胺對(duì)肺腺癌A549細(xì)胞增殖的抑制率呈時(shí)間依賴關(guān)系,作用24小時(shí)時(shí),尼克酰胺對(duì)肺腺癌A549細(xì)胞增殖的抑制率呈濃度依賴關(guān)系。隨著尼克酰胺濃度和作用時(shí)間的增加,肺腺癌A549細(xì)胞凋亡率明顯增加,并呈劑量及時(shí)間依賴關(guān)系。尼克酰胺組細(xì)胞遷移能力明顯弱于對(duì)照組,差異有顯著統(tǒng)計(jì)學(xué)意義。3.SIRT1影響肺腺癌生物學(xué)行為的可能的分子機(jī)制基因芯片mRNA表達(dá)譜檢測(cè)發(fā)現(xiàn),與肺腺癌A549細(xì)胞組比較毒黃素組共有745條差異基因表達(dá)上調(diào)倍值超過(guò)2倍,803條差異基因表達(dá)下調(diào)倍值超過(guò)2倍。與肺腺癌A549細(xì)胞組比較尼克酰胺組共有2505條差異基因表達(dá)上調(diào)倍值超過(guò)2倍,745條差異基因表達(dá)下調(diào)倍值超過(guò)2倍。分析同時(shí)在毒黃素組和尼克酰胺組顯著同向表達(dá)的差異基因,發(fā)現(xiàn)在兩組均表達(dá)上調(diào)的差異基因共159條,表達(dá)下調(diào)的差異基因共65條。由于SIRT1在肺腺癌中的生物學(xué)作用,進(jìn)一步分析發(fā)現(xiàn)凋亡通路中的Bcl-2相關(guān)轉(zhuǎn)錄因子1(BCLAF1)可能是SIRT1下游靶基因。通過(guò)TCGA數(shù)據(jù)庫(kù)分析發(fā)現(xiàn)SIRT1與BCLAF1均在肺腺癌組織共同表達(dá),并且兩者mRNA表達(dá)具有相關(guān)性。利用RT-PCR技術(shù),發(fā)現(xiàn)加入尼克酰胺共培養(yǎng)后,肺腺癌A549細(xì)胞的BCLAF1基因mRNA表達(dá)水平顯著升高。同時(shí),免疫印跡試驗(yàn)顯示BCLAF1蛋白水平也顯著升高。結(jié)論:本研究證實(shí)SIRT1在肺腺癌組織中的表達(dá)明顯高于相應(yīng)的癌旁正常組織,并且是肺腺癌預(yù)后不良的預(yù)測(cè)因子。SIRT1抑制劑可以抑制肺腺癌A549細(xì)胞增殖、遷移,促進(jìn)其凋亡。本研究利用人類全基因組寡核苷酸微陣列芯片,通過(guò)生物信息學(xué)技術(shù)篩選出差異基因:BCLAF1。并通過(guò)RT-PCR和免疫印跡法檢測(cè)發(fā)現(xiàn)SIRT1抑制劑能顯著升高肺腺癌A549細(xì)胞中BCLAF1基因mRNA和蛋白表達(dá)水平。因此,本研究提示SIRT1可能在肺腺癌的發(fā)生發(fā)展中扮演促癌基因的角色,其可能通過(guò)靶向調(diào)控BCLAF1參與肺腺癌的進(jìn)展過(guò)程。這為進(jìn)一步研究SIRT1的分子機(jī)制提供了理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R734.2

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