【作者】 王倩；

【导师】 周晓燕； 李小秋； 陆洪芬；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 硕士

【摘要】 DLBCL中SIRT1蛋白表达和基因异常及其对细胞生物学行为的影响背景弥漫性大B细胞淋巴瘤(Diffuse Large B Cell Lymphoma, DLBCL)是非霍奇金淋巴瘤中最常见且高度恶性的一种淋巴造血系统恶性肿瘤,占全部非霍奇金淋巴瘤的30-40%。在临床表现、形态学和遗传学表型上具有较大异质性,其分子机制尚不清楚,有待深入探讨。SIRT1基因(酵母沉默信息调节因子Sir2的同源基因)位于10q21.3,大小约33kb,编码一种重要的依赖于烟酰胺腺嘌呤二核苷酸(NAD+)的组蛋白去乙酰化酶,是由747个氨基酸构成的细胞生存调控蛋白。多项研究显示SIRT1通过去乙酰化组蛋白和一系列非组蛋白如P53,FOXO,Ku70,P300等从而发挥其延长细胞生命周期,抵御氧化应激损伤和保持基因组稳定的功能,因此又被称为长寿基因。但在恶性肿瘤中,SIRT1由于能抑制抑癌基因的转录活性等机制被认为是重要的肿瘤促进因子,FOXO(Forkhead box class0)转录因子是SIRT1的重要靶蛋白,受到SIRT1的去乙酰化调节改变其下游通道活性,从而影响细胞增殖凋亡状态。SIRT1的表达异常在前列腺癌、乳腺癌、皮肤癌等多种恶性肿瘤中均有相关报道,体内外实验证实抑制SIRT1表达或活性可诱导细胞生长阻滞或凋亡。关于SIRT1在DLBCL中的蛋白表达国外有1篇报道,但尚未见SIRT1基因状态及SIRT1在体外对DLBCL细胞株生物学行为影响的报道,国内也未见相关报道。目的观察SIRT1在DLBCL中的蛋白表达和基因状态及其临床病理学意义,分析两者的相关性,并进一步在体外观察慢病毒介导的SIRT1基因沉默对细胞生物学行为的影响。方法一、收集复旦大学附属肿瘤医院病理科1995年-2004年间本院活检为DLBCL的138例和淋巴组织反应性增生(Reactive Lymphoid Hyperplasia, RLH)的15例石蜡标本制成组织芯片,另增加6例RLH的单独制片,每例均由复旦大学附属肿瘤医院病理科两位病理医生复片,根据组织学形态和免疫组化特点(包括bcl-6、MUM1、CD10)进行诊断,诊断依据参照世界卫生组织最新分类标准。绝大多数病例都有年龄、性别、原发部位、临床分期、国际预后指数(International Prognostic Index, IPI)、血清乳酸脱氢酶(Lactate Dehydrogenase, LDH)值及组织学分型等临床特征数据。其中有67例有预后随访资料。随访时间2-120个月,截止至2009年12月份。二、采用免疫组织EnVision两步法,应用抗人SIRT1和FOX03a单克隆兔IgG抗体观察SIRT1和FOX03a在DLBCL中的蛋白表达和分布特征及其临床病理意义,进一步分析两者之间存在的相关性及与预后的联系；三、选用含有SIRT1基因全长序列的细菌人工染色体(Bacterial Artificial Chromosome, BAC)质粒的大肠杆菌单克隆扩增后提取质粒,经验证后用缺口平移法荧光标记SIRT1基因的杂交探针,荧光原位杂交法(Fluorescence In Situ Hybridiazaition, FISH)检测DLBCL、RLH及DLBCL细胞株lyl和ly8中SIRT1的基因异常情况,并分析与其蛋白表达之间的关联。四、CCK8染色法检测DLBCL细胞株lyl和ly8的增殖情况,Western blotting检测随着细胞增殖SIRT1和FOX03a蛋白含量的变化。利用RNAi和慢病毒转基因技术,构建pMagic4.0-SIRT1质粒,使它和pRsv-REV, pMDlg-pRRE,PMD2G组成慢病毒四质粒转染系统,共转染293T细胞,生产EGFP-SIRT1病毒。相应构建EGFP-NC空载体病毒作为阴性对照。用EGFP-NC、EGFP-SIRT1病毒分别感染lyl和ly8细胞,获取混合克隆,采用流式细胞仪筛选稳定表达EGFP-SIRT1、EGFP-NC的克隆,Western blotting检测SIRT1蛋白量判定干扰效率,比较分析SIRT1干扰组与对照组lyl和ly8的生物学行为的改变,采用CCK8染色法测定细胞增殖能力、流式细胞仪检测细胞周期和凋亡,以及用western blotting检测FOX03a,凋亡相关蛋白Caspase-3、Caspase-8和Caspase-9的蛋白含量变化。结果一、DLBCL的临床特征：该组DLBCL病例的发病年龄9-85岁,中位年龄47岁,其中男性74例,女性63例。59岁及以下的80例,59岁以上56例。原发于结内86例,结外51例,其中原发于胃肠30例。临床分期Ⅰ期30例,Ⅱ期42例,Ⅲ期27例,Ⅳ期6例。国际预后指数(International Prognostic Index, IPI)0-2分74例,大于2分的19例。LDH水平正常的47例,升高的35例。根据Hans等人的TMA判断法,将DLBCL分为GCB(Germinal center B-cell)51例(37%),non-GCB(Nongerminal center B-cell)87例(63%)。截止到2009.12.10,有随访资料的67例DLBCL患者中25例死亡,最长的存活期至今有10年。二、免疫组织化学结果：1. SIRT1在RLH中的阳性率为30% (6/20),明显低于在DLBCL中的阳性率67.2% (92/137)(P＜0.01)；2. FOX03a在RLH中主要在套区和套间增殖较不活跃的细胞中阳性表达,而生发中心的阳性细胞数少,表达较弱,FOX03a的总阳性率为90%(18/20)。在DLBCL中阳性细胞呈均—分布,总阳性率为81.7%(107/131)。两者无显著统计学意义(P＞0.05)；3.FOX03a在RLH中的强阳(+++)率为50%(10/20),在DLBCL中的强阳(+++)率为8.4%(11/131),两者具有显著统计学差异(P＜0.01)；4. SIRT1和FOX03a蛋白在RLH中的表达无关(P>0.05)；在DLBCL中两者表达呈正相关(x2=7.316,γ=0.237,P<0.01)：5. SIRT1在原发于胃肠的结外DLBCL中比其它结外或结内原发的DLBCL的蛋白表达率明显增高(P＜0.05),而与患者的年龄、性别、组织学分型、临床分期等其他临床特征均无关(P＞0.05)；6. SIRT1蛋白表达与DLBCL的预后未见明显相关(P＞0.05)。三、FISH结果：1.一共有127例DLBCL存在FISH信号,3例有SIRT1基因扩增,阳性率2.4%,33例为SIRT1基因低倍体(3-4倍体),占26.0%。6例为SIRT1基因高倍体(5倍及以上),占4.7%,即共有42例发生SIRT1基因数目异常(包括基因扩增或基因多倍体),约占所有DLBCL的33.1%；2. DLBCL中SIRT1基因数目异常和其蛋白表达之间呈正相关(γ=0.281,P=-0.001)；3.15例RLH中存在FISH信号,均未发现有SIRT1基因扩增或多倍体的现象；4.lyl和ly8细胞中均存在FISH信号,少于5%的细胞中见有SIRT1基因多倍体。四、体外研究的结果：1. SIRT1在DLBCL细胞株lyl和ly8传代后增殖早期表达水平较高,增殖后期表达水平降低；2.慢病毒EGFP-SIRT1转染后稳转细胞的筛选使lyl和l y8中SRIT1蛋白表达率相对于阴性对照分别下降78%和75%：3. SIRT1基因沉默对DLBCL细胞株lyl和ly8的周期和增殖能力未见有明显影响；4. SIRT1基因沉默可诱导DLBCL细胞株lyl和ly8凋亡,并上调细胞凋亡相关因子caspase3、caspase8和9,而FOX03a蛋白表达水平未见有明显改变。结论一、SIRT1在DLBCL中的蛋白表达阳性率要明显高于RLH,提示SIRT1参与了DLBCL的发生机制；二、FOX03a在DLBCL中的蛋白表达强阳性率要明显低于在RLH中,提示FOX03a潜在的抑癌作用；三、SIRT1和FOX03a的蛋白表达在DLBCL中正相关,在RLH中则没有关联,提示FOX03a表达和功能的调控可能是SIRT1的重要促癌机制之一；四、胃肠原发的DLBCL中SIRT1蛋白表达较高,提示SIRT1可能是这部分DLBCL发生的主要原因；五、DLBCL中SIRT1基因扩增十分少见而基因多倍体较常见,且与其蛋白表达正相关,提示SIRT1的基因多倍体是DLBCL中SIRT1蛋白表达增高的主要机制；六、SIRT1基因沉默可诱导DLBCL细胞凋亡,并同时介导内外源性的细胞凋亡途径。提示SIRT1通过抑制肿瘤细胞凋亡参与了DLBCL的发生机制。更多还原

【Abstract】 SIRT1 protein expression and gene status in DLBCL and its effects on biologic behaviour of DLBCL cell linesBackground:Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B lymphocytes accounted for 30%-40% ofNon-Hodgkin lymphoma. DLBCL is a heterogeneous tumor, clinically, morphologically and genetically. The molecular mechanisms are largely unknown and need to be further investigated. SIRT1 gene, the closest homologue of the yeast silent information regulator-2(Sir2), is 33kb and located on human chromosome 10q21.3. It encoded a nicotinamide adenine dinucleotid(NAD+)-dependent deacetylase which is a cellular growth-regulated protein composed of 747 amino acids. It had been shown to deacetylate a wide range of histone substrates and non-histone substrates including P53, FOXO family proteins, DNA repair protein Ku70, nuclear factorκB. SIRT1 was called longevity gene as it had broad biological functions in growth regulation, stress response and maintenance of genomic instability. However, the antiapoptotic effects of SIRT1 may paradoxically increase the risk of cancer. Transcription factor FOXO3a was an important target protein of SIRT1 which have effects on cellular proliferation and apoptosis. All of the functions may contribute to the development of cancer. SIRT1 was shown to highly express in a number of tumour types such as prostate cancer, skin cancer, breast cancer and so on. And targeted inhibition of SIRT1 in vitro and in vivo can effectively limit cancer progression and induce apoptosis. However, in DLBCL there was still rare both at home and abroad.Objective:To observe SIRT1 expression and gene status in DLBCL and to investigate the relationship with their clinicopathologic significance. To study the effects of lentivirus-mediated stable RNAi targeting SIRT1 gene on cellular biologic behavior in DLBCLcell lines.Methods:1. A306 dot tissue microarray consisting of 138 cases of DLBCL and 20 cases of RLH was made. All the paraffin-embedded tissues were retrieved from the files of the Department of Pathololy, Cancer Hospital, Fudan University between1995 and 2004 and all the histo logical slides were reviewed for confirmation of pathologic diagnosis by two professional pathologists. The diagnostic criteria refers to“WHO Classification:Tumors of Haematopoietic and Lymphoid Tissues(2008)”. The immunophenotypic and clinical features were collected and analyzed.67 patients were followed up for 2-120 months until December,2009.2. Immunohistochemical analyses of SIRT1 and FOXO3a were performed in DLBCL compared withRLH. The relationship between SIRT1 and FOXO3a expression and their clinicopathologic significance were analyzed respectively.3. The locations of BAC (RP11-44L18) included whole SIRT1 gene region were compiled from information archived by the UCSC and the NCBI. Probes were labeled by nick translation with Red-dUTP. The copy number of SIRT1 and CEP-10 were evaluated by fluorescence in situ hybridization (FISH) in the tissue array and lyl, ly8 DLCBCL cells. The correlation between SIRT1 gene copy number and protein expression was analyzed.4. Two DLBCLcell lines, lyl and ly8 were maintained in10%FBS culture medium. Cell Counting Kit-8(CCK-8) was used to detect the cell proliferation status. The expression of SIRT1 and FOXO3a were detected by Westernblotting. The relation between proteins expression and cell proliferation was analyzed respectively. Plasmids pMagic4.0-SIRT1 was constructed, which was combined with pRsv-REV, pMDlg-pRRE and pMD2G to constitute vector system of four plasmids. The lentiviral vector system was transfected into 293T cell to produce EGFP-SIRT1 lentivirus. EGFP-NClentivirus was also produced as a negative control. Lyl and ly8 cells were infected with both EGFP-NC and EGFP-SIRT1 lentivirus. Flow cytometry(FCM) were used to pick clones stably expressing EGFP-NC and EGFP-SIRT1 fragment. SIRT1 protein level was detected by western blotting. CCK-8 was used to assess the cell proliferation status. FCM was used to analyze cell cycle and apoptosis. Western blotting was used to detect FOXO3a, caspase-3, caspase-8 and caspase-9 which might relevant to cell apoptosis.Results:1. Clinical and Morphological features of DLBCL:there are 71 males and 63 females(M:F=1.1:1). The age ranged from9 to 85 years with median age of55 years.80 cases were younger and 56 cases were older than 59 years.86 cases were had at least on lymph node involved, and 51 were extranodal DLBCL. Of them,30 cases were in the gastroenteric tract de nevo. Patients were divided into 1(30),11(42),Ⅲ(27) and IV(6) according to their clinical stages.74 cases were 0-2 scores of the international prognostic index (IPI) and 19 cases were IPI scores of 3-4. There are 47 patients with normal serum LDH and 35 with high LDH. With the TMA algorithm described by Hans et al, 51 (37%) patients had GCB type and 87 patients(63%) had non-GCB types.of DLBCL.2. The results of immunohistochemistry:(1) The positive rate of SIRT1 expression in DLBCL(67.2%) was significantly higher than that in RLH(33.1%) (P<0.01). (2) In RLH, FOXO3a was charactistically expressed in the quiescent cells of the follicular mantle zone and in interfollicular lymphocytes, and only rare cells in the germinal center(GC) were positive. The possitive rate was 90%. But in DLBCL, FOXO3a was uniformly expressed with possitive rate of 81.7%. (3) No difference ofFOXO3a expression was found between RLH and DLBCL, but the strong positive expression rates of FOXO3a in RLH and DLBCL were significantly different(P<0.01). (4) No significant correlation was shown between SIRT1 and FOXO3a expression in RLH. However in DLBCL, SIRT1 and FOXO3a were significantly positively correlated (x 2=7.316,γ=0.237, P<O.01). (5) SIRT1 expression in patients with de novo gastrointestinal DLBCL was significantly higher than that in other extranodal or nodal DLBCL. (6) No statistical significant correlation was found between SIRT1 expression and sex, age, international prognostic index(IPI), serum LDH or pathological classification(P>0.05). (7) No relationship was found between SIRT1 and prognosis of DLBCL(P>0.05).3. The results of FISH detection:(1) 3 of the 127 DLBCL cases(2.4%) showed SIRT1 gene amplification. And 33 tetrasomy or trisomy(26.0%) and 6 polysomy cases(4.7%) were found. Overall,42 out of 127 DLBCL cases demonstrated SIRT1 gene alteration, either gene copy number increase or amplification, accounts for 33.1%. (2) SIRT1 gene alteration was significantly correlated with SIRT1 protein expression(γ=0.281, P=0.001). (3) No SIRT1 gene alteration was found in 15 RLH. (4) Less than 5% cells showed SIRT1 gene copy number increase in DLBCL cell lines lyl and ly8.4. The results of In Vitro study:(1) SIRT1 expression was much higher at the early phase after transfer of culture of lyl and ly8 cells and decreased at the later phase. (2) Lyl and ly8 cells stably expressing EGFP and EGFP-SIRT1 fragment were successfully picked. SIRT1 contents were obviously decreased by 78%in ly1 cell and 75% in ly8 cell(P<0.05). (3) The results of growth ability by CCK-8 method and cell cycle by flow cytometry showed no significant difference in all these cells. (4) Apoptosis by flow cytometry show significant difference by the effect of SIRT1 gene stably-silencing both in lyl and ly8 cells(P<0.05). (5) SIRT1 silencing up-regulated caspase-3,8 and 9 level in ly8 cell, but had no effect on FOXO3a level in all of the cells. Conclusions:1. SIRT1 expression can be observed more often in DLBCLthan in RLH. SIRT1 may play an important role in the tumorigenesis of DLBCL.2. FOXO3a expression was stronger in RLH than in DLBCLwhich suggest that FOXO3a gene is a putative tumor suppressor gene3. SIRT1 expression are positively correlated with nuclear expression of FOXO3a in overall DLBCL. SIRT1 may be involved in the development of DLBCL though the regulation of FOXO3a expression and its functioa4. SIRT1 may play an important role in de nevo gasterointestinal DLBCL.5. SIRT1 amplification was rare in DLBCL while gene copy number increase was very common, accounts for 1/3 cases. SIRT1 gene alteration significantly contributes to protein expression of SIRT1 in DLBCL.6. SIRT1 silencing induces apoptosis in DLBCL cells, and both extrinsic and intrinsic pathway maybe required which suggests that SIRT1 had anti-apoptosis effects in DLBCL.更多还原