【作者】 毛海婷；

【导师】 张利宁；

【作者基本信息】 山东大学 ， 免疫学， 2010， 博士

【摘要】 研究目的乳腺癌是严重威胁女性健康的恶性肿瘤,也是一种基因学上异质性的疾病,这种基因的异常又决定着肿瘤的特性,同时也是决定其临床进展行为的基础。明确决定乳腺癌进展、转移的关键基因,将有助于设计有效的预防和治疗药物。如果决定复发和转移危险性的关键基因在原发肿瘤诊断时即可得到评定,将有助于设计个体化的预防和治疗方案。所以,发现决定乳腺癌发生与进展的关键基因已经成为世界范围内急需攻克的科研难题。目前有关乳腺癌基因发病机制的研究多聚焦于家族性乳腺癌以及与其发病密切相关的BRCA1 (Breast cancer 1, early onset)和BRCA2 (Breast cancer 2, early onset)基因缺陷,但约90%罹患乳腺癌的女性是由与BRCA1或BRCA2基因缺失无关的非家族性乳腺癌发展而来,其基因发病机制尚不明确。2002年,美国研究者发现了一种新的乳腺癌抑制基因DBC2 (deleted in breast cancer 2),亦命名为RhoBTB2,并推测DBC2在大多数乳腺癌中表达缺失或处于失活状态,更重要的是,它是与90%的散发性即非家族性乳腺癌密切相关的少数基因之一。初步的研究显示,DBC2基因在多种乳腺癌细胞系和肺癌细胞系中表达缺失。遗传学分析发现,DBC2基因在近10%的乳腺癌中发生了纯合性缺失或点突变等明显的基因改变,而同样位于这一区域的、在正常乳腺组织中表达的基因DBC1及TNFRSF10B均未发生明显的突变,因此DBC2被视为乳腺癌的候选肿瘤抑制基因。虽然DBC2的抑癌基因作用得到了初步认识,但其详细作用机制目前尚不完全清楚。DBC2基因的发现及研究对于阐明散发性乳腺癌及其它肿瘤的发病机制有着极其重要的意义。目前,DBC2的基因定位、蛋白结构及组织表达模式已基本明确,但其作为抑癌基因的功能及作用机制的研究很少,也存在很大的局限性。虽然DBC2最早是从乳腺癌中克隆出来的抑癌基因,然而迄今为止的研究对DBC2在肿瘤组织,尤其是散发性乳腺癌组织中的表达状况仍然一无所知。DBC2通过何种方式发挥抑癌基因的作用?其表达变化能从哪些方面引起细胞功能学的改变?DBC2在肿瘤组织中的表达缺失与乳腺肿瘤的发生、发展、预后及临床病理之间存在怎样的关联?尚不明确。而另一方面,由于DBC2参与细胞骨架的形成及微管介导的蛋白转运,其在肿瘤病理条件下是否能影响肿瘤细胞的迁移、侵袭和转移?所有这些均是本研究的主要目的和我们需要进一步探讨的焦点问题。本论文从临床病例到体外细胞模型,系统地研究了DBC2在乳腺癌的发生发展中的作用、功能机制及其与患者预后的关系：1.抑癌基因DBC2在非家族性乳腺癌中的缺失表达状况及与患者临床病理的相关性；2.DBC2在乳腺癌患者预后转归中的作用；3.构建DBC2真核表达载体和DBC2稳定转染的乳腺癌细胞系,作为基因功能研究的模型；4.从体外细胞水平研究DBC2对乳腺癌细胞增殖、克隆形成、细胞周期、凋亡及侵袭转移的影响。研究方法一.抑癌基因DBC2在非家族性乳腺癌中的表达状况及其临床意义1.病例资料所有病例标本均取自山东省肿瘤防治研究院及山东大学齐鲁医院收治的乳腺疾病患者,乳腺癌组60例,均为女性首诊可手术的散发性乳腺癌,术前未接受任何治疗。正常乳腺标本30例,取自乳腺良性病变旁正常腺体组织,均经术后病理证实为正常乳腺组织。乳腺癌组发病年龄28～70岁,中位发病年龄46岁。乳腺癌病例术后随访至2009年8月,平均随访48个月,失访4例。2.细胞系及组织标本人宫颈癌细胞系HeLa、人卵巢癌细胞系SKOV3、3AO、人膀胱癌细胞系BIU-87、人胃癌细胞系MGC803、人乳腺癌细胞系MCF-7、SKBR3、T-47D、人胚肺成纤维细胞系HELF-6。新鲜胎儿脑组织取自山东大学齐鲁医院妇产科自然流产胎儿。3. RT-PCR法检测组织标本及细胞系中DBC2 mRNA的表达状况通过RT-PCR法检测8种肿瘤细胞系、正常胚胎组织细胞、60例乳腺癌组织及30例正常乳腺组织中DBC2在mRNA水平的表达状况。4.免疫组化法检测组织中DBC2蛋白水平的表达应用免疫组化方法检测60例乳腺癌组织及30例正常乳腺组织中DBC2在蛋白水平的表达状况。5.统计学方法乳腺癌组和正常乳腺对照组以及不同临床病理类型的肿瘤患者之间DBC2缺失表达率的不同采用Chi-square检验。Kaplan-Meier method用于绘制生存曲线,Log-rank和Gehan-Wilcoxon检验用于分析DBC2表达与肿瘤患者生存时间的关系,Cox比例风险回归用于多因素生存分析。二.DBC2真核表达载体的构建及阳性细胞系的筛选1.真核表达载体的构建：运用RT-PCR技术扩增DBC2基因DNA片断,经纯化回收,将DBC2基因片断与克隆载体pMD19-T simple vector连接,转化至感受态E.coli JM109大肠杆菌中,并进行纯化、酶切鉴定及测序。将测序正确的目的基因DBC2亚克隆至真核表达载体pEGFP-N1中,构建成重组真核表达质粒pEGFP-N1-DBC2。2.阳性细胞系的筛选：利用脂质体法将质粒转染入T-47D细胞：转染分以下三组进行：①pEGFP-N1-DBC2转染组；②pEGFP-N1空质粒转染组；③单纯脂质体对照组,通过有限稀释法筛选阳性单克隆细胞,并对稳定转染的细胞系进行RT-PCR及Western Blot鉴定。三.外源性DBC2过表达对乳腺癌细胞恶性行为的影响1.MTT法细胞增殖抑制的检测：取对数生长期的不同处理组T-47D细胞,调细胞浓度为3×104/ml,分别接种于五块相同的96孔板,置于培养箱中分别培养0、1、2、3、4天,酶标仪检测A值,绘制动态生长曲线。2.克隆形成实验：将不同处理组的T-47D细胞分别接种于6孔板,200细胞／孔,置于37℃、5%CO2孵箱中静置培养2-3周,甲醇固定细胞及Giemsa染色,显微镜下计数≥50个细胞的克隆数,计算克隆形成率。3.流式细胞术检测细胞周期：收集不同处理组的T-47D细胞(～106细胞),70%冷乙醇4℃固定过夜,加入PI染液避光染色30min,上机检测分析。4.HE染色法检测细胞凋亡：将细胞分别接种于铺有玻片的6孔板,置于37℃、5%CO2孵箱培养48h,HE常规染色。5.流式细胞术Annexin V-APC/PI法检测细胞凋亡百分率：收集不同处理组的T-47D细胞,4℃预冷的PBS洗两次,加入5μl Annexin V-APC和10μl PI溶液,室温避光孵育15 min,上机检测。6.细胞侵袭能力的检测：将含10%FBS的DMEM培养基加到侵袭小室的下室,细胞悬液加入侵袭小室的上室,37℃、5%CO2孵箱中孵育72h,拭去上室中非侵袭细胞及ECMatrix胶,染色及显微镜下观察和拍照。每孔加入10%醋酸150μl抽提细胞,酶标仪上检测OD570光密度值。7.细胞迁移能力的检测：除上室中未铺ECMatrix胶及培养时间为8h外,余步骤同侵袭实验。结果一.抑癌基因DBC2在非家族性乳腺癌中的表达状况及其临床意义1.DBC2在胚胎组织、细胞及不同肿瘤细胞系中的表达为了探讨DBC2基因在肿瘤发生和发展中的作用,我们首先通过RT-PCR法检测了该基因在8种不同的人肿瘤细胞系及正常人胚胎组织和细胞中的表达状况,结果显示,DBC2不仅表达于正常的胎脑组织和人胚肺细胞,亦表达于乳腺腺癌(MCF-7、SKBR3)及宫颈癌(HeLa)、卵巢癌(SKOV3、3AO)、膀胱癌(BIU-87)、胃癌(MGC-803)等其它来源的肿瘤细胞系,但在乳腺导管癌细胞T-47D中表达缺失,提示DBC2可能与乳腺导管癌的发生有关。2.DBC2在正常乳腺及乳腺癌组织中的表达上述结果显示,在我们检测的所有肿瘤细胞系中,DBC2仅在部分乳腺癌细胞中表达缺失。为进一步探究DBC2在原发性乳腺癌组织中的缺失表达状况,本研究分别采用RT-PCR技术及免疫组化方法检测了60例乳腺癌组织和30例正常乳腺组织中DBC2的表达状况。(1)DBC2在正常乳腺及乳腺癌组织中mR_NA水平的表达检测RT-PCR结果显示,30例正常乳腺组织中有28例可检测到DBC2 mRNA水平的表达,而60例乳腺癌标本中有36例表达缺失,两组的缺失表达率有明显差异(6.7% versus 60.0%, P<0.001)。(2)DBC2在正常乳腺及乳腺癌组织中蛋白水平的表达检测基于RT-PCR的结果,我们进一步检测了DBC2蛋白水平的表达,免疫组化结果显示,DBC2表达于乳腺上皮细胞的胞浆中,阳性结果呈棕黄色染色。几乎所有正常乳腺组织均有表达(28／30),而乳腺癌组仅有24例可检测到DBC2的弱阳性表达,其表达水平明显低于正常乳腺组,其余36例标本均为阴性。这与RT-PCR的检测结果相吻合。3.DBC2的表达缺失与患者临床病理的相关性分析本研究中我们将乳腺癌组按照不同的临床病理资料进行分类,以期发现DBC2的表达缺失与患者临床病理因素之间的相关性。统计结果显示,DBC2的缺失表达与患者的发病年龄、孕激素受体(PR)的表达以及肿瘤的病理类型密切相关。年龄之50岁的患者与年龄<50岁的患者相比,DBC2的阴性率更高(90.0%versus 45.0%, P=0.000); PR阳性患者的DBC2缺失率也明显高于PR阴性的患者(72.2%versus 41.7%,P=0.018)。此外,不同肿瘤亚型DBC2的缺失率也不同,与浸润性小叶癌相比,DBC2的高频缺失更易发生在浸润性导管癌(28.6% versus71.4%,P=0.000)。这一结果与DBC2在不同肿瘤细胞系中的表达检测相一致,即DBC2缺失表达的乳腺癌细胞系T-47D来源于一位54岁、雌激素和孕激素受体均表达阳性的女性乳腺浸润性导管癌患者。但是DBC2的表达与患者的临床分期、腋窝淋巴结转移、雌激素受体(ER)以及HER2受体的表达无相关性。4.DBC2在乳腺癌中的表达状态与病人预后的关系本研究对56例乳腺癌病例(4例失访)进行了术后随访,并通过Kaplan-Meier方法和Log-rank检验将DBC2的表达与患者的术后生存率进行了评估,发现DBC2的表达与患者的长期生存百分率密切相关,DBC2阳性患者的生存率显著高于DBC2阴性患者(P<0.05)。通过对包括发病年龄、肿瘤病理类型、临床分期、腋窝淋巴结转移、ER、PR以及DBC2表达在内的多因素Cox回归分析显示,DBC2表达是乳腺癌患者生存的独立预后因素,DBC2表达缺失的乳腺癌患者预后不良(相对危险系数,0.090；95%可信区间：0.010-0.806：P=0.03)二.DBC2真核表达载体的构建及稳定细胞系的筛选和鉴定本研究选取DBC2表达阳性的人胚肺成纤维细胞系HELF-6作为DBC2基因的来源,通过RT-PCR方法获取该基因的DNA,经过测序、基因的亚克隆等步骤将目的基因与表达载体pEGFP-N1连接,构建了携带有绿色荧光蛋白和G418抗性的重组质粒,通过脂质体法转染入DBC2表达缺失的人乳腺癌细胞系T-47D,筛选出稳定转染的T-47D细胞,并通过RT-PCR和Western blot方法分别从基因和蛋白水平鉴定了目的基因的表达,为该基因的功能研究建立了良好的细胞模型。三.外源性DBC2过表达对乳腺癌细胞恶性行为的影响1.DBC2表达对乳腺癌细胞形态的影响未进行转染及空载体转染(Mock组)的乳腺癌细胞T-47D生长良好,细胞仍保持原有的生长形态。而DBC2过表达的乳腺癌细胞其数量和形态发生了明显变化：细胞数量减少,胞质丰富,胞质内颗粒增多,细胞多成群、成团排列,有向正常乳腺细胞分化的趋势。2.DBC2对乳腺癌细胞的生长抑制作用为了研究DBC2作为抑癌基因的功能,我们首先通过MTT法检测了DBC2对乳腺癌细胞生长的影响。0、1、2、3、4天五个时间点的细胞动态生长曲线显示,与未处理的T-47D细胞阴性对照组及Mock组相比,目的基因转染组的细胞生长从第二天开始明显减慢,并随生长时间的延长,差异更加显著(P<0.01),而Mock组的细胞生长未受影响。3.DBC2对乳腺癌细胞克隆形成的影响为了进一步评估DBC2表达对乳腺癌细胞增殖的影响,我们检测了目的基因转染后乳腺癌细胞克隆形成能力的改变。与正常细胞不同,肿瘤细胞具有在培养介质中形成克隆的倾向和能力,克隆样生长也是恶性肿瘤的基本特征之一。我们的研究发现,DBC2具有抑制乳腺癌细胞克隆形成能力的作用。过表达DBC2转染组T-47D细胞的克隆形成率及所形成的克隆大小均明显低于阴性对照组和空载体Mock组。与Mock组相比,其克隆形成率仅为26%±4.24%(P<0.01)。4.DBC2对乳腺癌细胞周期的影响上述实验结果表明,DBC2可通过抑制乳腺癌细胞的增殖和克隆形成能力而阻抑细胞的恶性生长。为探究这种生长抑制的机制,我们检测了DBC2对肿瘤细胞周期的影响。流式细胞检测结果显示,外源性DBC2的表达可显著抑制乳腺癌细胞周期的演进。与阴性对照及Mock组相比,DBC2转染组出现了明显的G0／G1期比例升高(分别为46.32%,53.92%和80.23%)及S期比例的降低(分别为46.91%,35.54%和11.36%)。这说明D.BC2对乳腺癌细胞的生长抑制作用是通过诱导细胞周期G1期阻滞,从而阻止细胞进入DNA合成期(S期)得以实现的。5.DBC2对乳腺癌细胞凋亡的影响本研究分别采用两种检测方法观察了DBC2对肿瘤细胞凋亡的影响。HE染色形态学观察发现,T-47D对照组及空载体组的乳腺癌细胞仍然保持原有的生长形状,细胞核完整,呈均匀蓝色或淡蓝色,胞浆淡红色。而外源性DBC2过表达的乳腺癌部分细胞变圆,与周围细胞脱离,细胞质密度增加,细胞核固缩、浓染,出现典型的凋亡小体。细胞经Annexin V-APC/PI染色后,流式细胞仪分析结果显示,外源性DBC2的表达可显著促进乳腺癌细胞的凋亡,凋亡率为22.07%±1.67%,明显高于T-47D对照组(8.31%±1.10%)(P＞0.001)和空载体组对照组(11.25%±2.59%)(P<0.01)。6.DBC2对乳腺癌细胞侵袭能力和迁移能力的影响侵袭和转移是恶性肿瘤最显著的生物学特性,肿瘤细胞的侵袭和转移过程涉及到多个阶段和环节,其中最为重要的是对细胞外基质的降解侵袭能力和迁移运动能力。本研究首先利用铺有细胞外基质(ECMatrix)的Transwell系统检测了DBC2对乳腺癌细胞侵袭转移能力的影响。72小时的细胞侵袭实验结果表明,与未转染的T-47D细胞和Mock对照组相比,过表达DBC2的肿瘤细胞侵袭能力无明显变化(分别为0.56±0.06,0.47±0.04和0.50±0.02,P>0.05)。同样,8小时的细胞迁移实验也证明,过表达DBC2的肿瘤细胞迁移能力与未转染的T-47D细胞及Mock组相比,其细胞运动和迁移能力无明显改变(分别为0.53±0.20,0.51±0.10,0.51±0.05,P>0.05)。这与本研究第一部分临床资料的分析结果相吻合,即乳腺癌患者DBC2的表达缺失与患者的临床分期及腋窝淋巴结转移无相关性。结论1.DBC2在散发性、非家族性乳腺癌组织中高频缺失,其基因的失活发生于转录水平。2.DBC2的表达缺失更易发生在50岁以上的绝经妇女和孕激素受体阳性的患者中；浸润性导管癌DBC2的缺失率更高。这说明DBC2可能受雌性激素的调节,其缺失表达在乳腺导管癌的发病机制中发挥重要作用。3.DBC2的缺失与乳腺癌患者的不良预后有关,是影响乳腺癌预后的独立因素。4.DBC2可通过抑制肿瘤细胞生长、降低克隆形成能力、诱导细胞周期G1期阻滞以及促进肿瘤细胞凋亡等方式发挥抑癌基因的作用,但其对肿瘤细胞的侵袭和转移能力无明显影响。创新性及意义1.本研究检测了乳腺癌组织中DBC2的表达缺失状况,并首次阐明DBC2的表达缺失与患者的临床病理及预后转归的相关性。2.本研究首次通过体外细胞功能实验从增殖、凋亡及侵袭转移等方面全面证实了DBC2作为抑癌基因的功能。3.本研究结果为DBC2的后续研究提出了新问题,为以DBC2为靶点的基因靶向治疗和防治策略提出了新思路。更多还原

【Abstract】 ObjectiveBreast cancer is the most common malignancy in women and also is a genetic disease. Like other human cancers, it is thought to occur as the result of progressive accumulation of genetic aberrations. Familial breast cancer is characterized by an inherited susceptibility to breast cancer on basis of an identified germline mutation in one allele of a high penetrance susceptibility gene, such as BRCA1, BRCA2, CHEK2, TP53 or PTEN. But in more than 90% of cases, breast cancers result from a serial stepwise accumulation of acquired and uncorrected mutations in somatic genes, without any germline mutation playing a role. Up to now, the genetic alterations that precede development of sporadic breast cancer are poorly understood. Recently, Hamaguchi and coworkers have used representational difference analysis to identify a novel tumor suppressor gene DBC2 (Deleted in Breast Cancer 2), also named as RhoBTB2 gene, which belongs to the Rho GTPase family and is located at chromosome 8p21 deleted in some breast and lung tumor cell lines.DBC2 protein is an atypical member of Rho GTPase family. Unlike typical Rho GTPases, distinctive functions of DBC2 have been implied due to their unique structure such as transcriptional regulation and protein degradation. Studies indicated that DBC2 was homozygously deleted in 3.5% of primary breast cancers, gene expression was ablated in about 50% of breast and lung cancer cell lines, and several somatic missense mutations in DBC2 were isolated from primary tumors and cancer cell lines. Furthermore, reintroduction of DBC2 into a breast cancer cell line lacking endogenous DBC2 expression led to growth arrest. Although DBC2 was initially cloned as a tumor suppressor gene in breast cancer, its tumor suppression mechanism, the influence to tumor cells function and the status of expression in human sporadic breast cancer tissues remain unclear.In the present study, we will approach the antitumor effect of DBC2 at the following aspects:1. The expression status of DBC2 in sporadic breast cancer and its clinical significance.2. Construction of eukaryotic recombinant vector of human DBC2 and establishment of its stable expression cell line.3. Overexpression of exogenous DBC2 gene in breast tumor cells and the function study of RhoBTB2.Methods 1. The expression status of tumor suppressor gene DBC2 in sporadic breastcancer and its clinical significance(1) Patients and normal donors Sixty unrelated Chinese women with sporadic breast cancer, ranging in age from 28 to 70 years old (median 46) and 30 unrelated age matched control women with benign breast disease participated in this study. Normal control tissues were resected aside from benign breast disease tissues. Breast cancers were proved from surgical, pathological and clinical information obtained from their medical files. Any patient who had received chemotherapy or radiotherapy before obtaining specimens was excluded from this study. Survival was assessed in 56 patients as of August,2009 (4 Missing follow-up).(2) Cell lines and normal tissues samples HeLa:cervix carcinoma cell line; SKOV3,3AO:ovarian carcinoma cell line; BIU-87:bladder carcinoma cell line; MGC803:gastric carcinoma cell line; MCF-7, SKBR3:breast adenocarcinoma cell line; T-47D:breast ductal epithelial carcinoma cell line; HELF:human embryo lung fibroblasts cell line. Fetal brain tissue came from fetus of spontaneous abortion in Qi Lu Hospital, Shandong University.(3) The tissue samples from breast cancer patients and normal controls and cell samples from cell lines were collected and reverse transcription PCR (RT-PCR) was performed to detect the DBC2 mRNA. (4) The protein expression in 60 cases of breast cancer specimens and 30 cases of normal controls was detected using immunohistochemical staining (avidin-biotin-peroxidase complex method).(5) The relationship between the DBC2 expression and the pathological characteristics was analyzed by statistics. Cumulative survival time was calculated by the Kaplan-Meier method and curves were analyzed by the Log-rank and Gehan-Wilcoxon test. Multivariate survival analysis was performed using the Cox proportional hazard regression model.2. Construction of eukaryotic recombinant vector of human DBC2 and establishment of its stable expression cell line(1) Construction of recombinant vector of DBC2:Coding DNA sequence of human DBC2 was amplified by RT-PCR from HELF-6 cell line and was cloned into pMD19-T simple vector and then was transformed into competent E.coli JM109. The entire insert was sequenced from both directions by the method of Primer Walking. Full-length DNA of DBC2 was cloned into EcoRl-BamHl sites of pEGFP-N1 plasmid to get a recombinant eukaryotic expression vector named pEGFP-N1-DBC2.(2) Transformation:T-47D cells were transformed with recombinant plasmid by liposome LipofectAMINE 2000 Reagent following the manufacturer’s instructions. Three groups were included in the transformation:①pEGFP-N1-DBC2 group;②pEGFP-N1 group;③liposome control group.(3) Screening of stable transformant clones:Stable transformants were selected by limiting dilution analysis and using G418 at a higher level than the minimum deadly concentration. The DBC2 expression of stable transformant clones was identified by RT-PCR and Western Blot.3. Overexpression of exogenous DBC2 gene in breast tumor cells and its function study of tumor suppressor gene DBC2To determine the effect of DBC2 on the growth, cell cycle, colony formation, apoptosis, invasion and metastasis of breast tumor cells, the recombinant plasmid pEGFP-N1-DBC2 carrying the full-length DBC2 cDNA was transfected into DBC2-negative breast tumor cells T-47D. (1) Cell proliferation was evaluated by MTT assayT-47D cells from different treated groups were incubated at 3×104/ml in 96-well flat bottom plates in DMEM medium supplemented with 10% FBS. Five identical plates were prepared for different time point assays (0,1,2,3,4 day, respectively). In each plate,5g/L MTT was added to cells for the last 4 h. Absorbance was determined with an enzyme-linked immunosorbent assay reader using 570 nm as test wavelength and 630 nm as reference wavelength.(2) Colony formation assayT-47D cells from different treated groups were plated in 6-well plates with a density of 200 cells per well in the presence of G418 for 2-3 weeks. The colonies were fixed in methanol and stained with Giemsa staining solution. The number of colonies with≥50 cells was counted and colony forming efficiency was calculated.(3) The cell cycle was detected by flow cytometric analysisT-47D cells from different treated groups (～106) were washed twice with cold PBS and were fixed in 70% cold ethanol for overnight at 4℃. Then, the cells were incubated with PI staining solution for 30 min in the dark. The stained cells were analyzed with a FACS Calibur flow cytometer using the Cell Quest software.(4) The apoptotic morphology detection by Hematoxylin-Eosin (HE) stainingT-47D cells were incubated in 6-well plate paved with grass slides at its bottom for 48 h at 37℃in 5% CO2. Then the slides were washed with PBS and were stained by HE method.(5) The apoptosis ratio was detected by flow cytometric analysisT-47D cells from different treated groups were collected and were washed with cold PBS. Five microliter Annexin V-APC and 10μl PI solution were added to cells suspension and incubated for 15 min in the dark. The stained cells were analyzed with flow cytometer.(6) Cell invasion assayCell invasion analysis was performed using a 24-well transwell chamber. Tumor cells were incubated in DMEM medium with serum free and were seeded in the upper chamber with an 8μm pore size insert precoated with ECMatrix in the 24-well plate and cultured for 72 h. Cells were allowed to migrate towards medium containing 10% FBS in the bottom chamber. The non-migratory cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were stained with 0.1% crystal violet. The crystal violet dye was eluted by 10% acetic acid and the absorbance was determined with an enzyme-linked immunosorbent assay reader using 570 nm as test wavelength.(7) Cell migration assayThe test method was the same as cell invasion assay except that there was not ECMatrix in the upper chamber and incubation time was 8 h.Results1. The expression status of tumor suppressor gene DBC2 in sporadic breast cancer and its clinical significance(1) Expression of DBC2 in embryon and different cancer cell linesDBC2 was expressed in human fetal brain tissue, human embryonic lung fibroblasts cells, uterine cervical carcinoma, ovarian carcinoma, bladder carcinoma, gastric carcinoma, breast adenocarcinoma cells MCF-7 and SKBR3, was not expressed in T-47D, a breast ductal epithelial carcinoma cell line.(2) Detection of DBC2 mRNA in breast cancer tissues and normal controlsThe DBC2 expression at RNA level in 60 breast cancer tissues and 30 normal breast samples was analyzed by RT-PCR. DBC2 mRNA expression was amplified in 28 of 30 normal breast samples, in contrast, most of breast cancer lacked DBC2. In 60 breast cancer samples,36 lost DBC2 mRNA expression. There was a significant difference of negative ratio between these two groups (6.7% versus 60.0%, P<0.001).(3) Expression analysis of DBC2 in breast cancer tissue and normal controlsWe further detected the expression of DBC2 at protein level by immunohistochemistry (IHC) in breast cancer tissue samples and normal breast tissue. Peroxidase staining revealed cytoplasmic expression in almost all of breast ductal epithelial cells samples from normal breast tissue (28/30). Whereas DBC2 was only weakly expressed in part of breast cancer samples or was not expressed in the most of breast cancer samples (36/60). These results were consistent with the RT-PCR analysis.(4) Association of loss of DBC2 expression in breast cancer tissues and clinicopathologic informationResults revealed an association between age of onset, progesterone receptor (PR) expression, tumor type and loss expression of DBC2. There was significantly higher negative ratio of DBC2 in patients of age≥50 years old than that of in patients of age <50 years old (90.0% versus 45.0%, P=0.000). Meanwhile, there was an increase of DBC2 loss expression ratio among patients with positive PR compared to those with negative PR (72.2% versus 41.7%,P=0.018). In this study, we also found that frequent loss of DBC2 occurred easily in ductal carcinoma as opposed to lobular carcinoma (71.4% versus 28.6%, P=0.000). This result agreed with the expression analysis of DBC2 in different cancer cell lines. But there was no correlation between loss of DBC2 expression and clinical stages, axillary lymph nodes metastasis, estrogen receptor (ER) and HER2 expression.(5) Prognostic value of DBC2 expression for patients with breast cancerTo investigate the association of DBC2 expression with patient survival, the survival data from 56 patients with breast cancer (4 missing follow-up) were assessed. In univariate analysis, the expression ratio of DBC2 significantly correlated with the long-term survival rate of patients. The survival rate of patients with DBC2-positive breast cancer was significantly higher than that of patients with DBC2-negative breast cancer (P=0.02). Results from the multivariate analysis showed that DBC2 expression was a significant prognostic factor and correlated with a better clinical outcome (relative risk,0.090; 95% confidence interval,0.010-0.806; P= 0.03).2. Construction of eukaryotic expression vector of DBC2 and establishment of its stable expression cell lineFull-length human DBC2 was obtained from human fetal lung cell line HELF-6 with the addition of EcoRl restriction endonuclease recognition sites at its 5’end and BamHl site at the 3’end. The recombinant eukaryotic expression vector with green fluorescent protein, pEGFP-N1-DBC2, was successfully constructed and identified by digestion with restriction enzyme. DBC2 gene was introduced into T-47D cells and stable transformants were obtained by screening with G418 and were identified by RT-PCR and Western blot. This provided a perfect cell model for the functional analysis of DBC2.3. Overexpression of exogenous DBC2 gene in breast tumor cells and its functional study(1) The morphological changes of T-47D cells with DBC2 expressionThe overexpression of DBC2 caused the morphological changes of T-47D cells compared with T-47D negative controls and empty vector transfected (Mock) group. There was decreased numbers of cells, abundant cytoplasm, increased cytoplasmic particles and cells arranged in groups in breast tumor cells transfected with DBC2.(2) Overexpression of DBC2 in breast tumor cells inhibites proliferationThe wild type DBC2 expression in T-47D cells significantly inhibited their growth compared with untreated negative cell control and mock group (P<0.01).(3) Overexpression of DBC2 in breast tumor cells prevents colony formationBreast tumor cells, unlike normal breast cells, have the tendency to form colonies in the culture. The overexpression of DBC2 significantly diminished the capacity of breast tumor cells to form colonies. Compared with mock group, the expression of DBC2 caused reduction in the number of colonies formed by T-47D cells (26%±4.24%, P<0.01).(4) Overexpression of DBC2 in breast tumor cells arrestes the cell cycleCell cycle analysis showed that there were significant differences in proportions of G0/G1 and S phase between T-47D cells transfected with DBC2 and their control groups. Expression of DBC2 in breast tumor cells increased the proportion of G0/G1 phase (80.23% vs 46.32% T-47D controls and 53.92%mock controls) and reduced the proportion of S phase (11.36% vs 46.91% T-47D controls and 35.54% mock controls) and thereby prevented tumor cells entering DNA synthesis phase. (5) Overexpression of DBC2 in breast tumor cells promotes the apoptosisResults from Annexin V-APC/PI analysis revealed that T-47D cells transfected with DBC2 underwent obvious apoptosis (22.07%±1.67%) than negative cell controls (8.31%±1.10%) and mock (11.25%±2.59%) (P< 0.01). HE morphological staining also showed that there were clearly apoptosis morphological changes in breast tumor cancer cells of DBC2 overexpression, such as cells becoming round, cytoplasmic density increasing, cell nucleus shrinking, concentrated dye and typical apoptotic bodies.(6) Effect of DBC2 on invasion and migration ability of breast tumor cellsTo investigate the effect of DBC2 on breast tumor cells invasion and metastasis ability, the transwell system precoated with ECMatrix was used in this study. Our results showed that overexpression of DBC2 did not affect the invasion ability of breast tumor cells compared with negative cell controls and mock controls, (0.50±0.02 and 0.56±0.06,0.47±0.04, respectively, P>0.05). The effect of DBC2 on breast tumor cells migration ability also be evaluated by transwell assay. The overexpression of DBC2 did not affect the migration ability of breast tumor cells compared with negative cell controls and mock controls, (0.51±0.05 and 0.53±0.20, 0.51±0.10, respectively, P>0.05).Conclusion1. High frequently loss of DBC2 generally exist in breast cancer tissues and the inactivation of DBC2 may occur at the transcription level. The loss of DBC2 expression more frequently occur in postmenopausal patients with age≥50 and in patients with PR-positive expression. Infiltrative ductal carcinoma subtype has higher negative ratio of DBC2 than that of infiltrative lobular carcinoma subtype. Furthermore, DBC2 expression may be controlled by female hormones and it functions as tumor suppressor in a tissue specific manner.2. DBC2 expression can serve as a considerable factor for prognosis of sporadic breast cancer.3. DBC2 plays antitumor roles by inhibiting proliferation, preventing colony formation, arresting cell cycle and promoting the apoptosis of tumor cells. But DBC2 does not affect the invasion and migration ability of breast tumor cells. Originality1. We demonstrate, for the first time, that loss of DBC2 expression is a frequently event in human breast cancer and that frequently loss of DBC2 significantly correlates with the clinicopathology, outcome and prognosis of breast cancer patients.2. It is the first time, we thoroughly reveal the function of DBC2 as a tumor suppressor by proliferation, apoptosis, invasion and metastasis in cell functional study.3. Our finding raises new issues for the follow-up study of the DBC2 gene and put forward new ideas for gene targeted therapy of DBC2.更多还原