抑癌基因Numb在恶性胸膜间皮瘤中表达的临床意义及其功能研究
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摘要
研究背景和目的
恶性胸膜间皮瘤(malignant pleural mesothelioma, MPM)是一种少见的胸膜部侵袭性肿瘤,预后极差。它的发病通常与接触石棉有关,尽管自19世纪70年代以来石棉的使用广受限制,但接触后的长期潜伏期使间皮瘤的发病率正持续上升。预计在2010-2020年间,工业发达国家恶性间皮瘤的死亡率将达到高峰。根据组织形态学分类,MPM可分为上皮型、肉瘤样型(纤维型)和混合型三种类型,以上皮型多见。本病传统治疗效果差,确诊后中位生存期约为10-12个月。因此,对MPM有效的诊疗措施亟待研究。近些年来,基因靶向治疗在肿瘤领域的成功应用为MPM寻求有效的治疗方法奠定了基础。
Numb是生物体内最重要的细胞命运决定因子之一,被认为参与多种恶性肿瘤的形成,被认为是颇具希望的抑癌基因。Numb是一种膜相关蛋白,其结构包含一个磷酸酪氨酸结合域(phosphotyrosine binding, PTB)和一个富脯氨酸域(proline rich region, PRR);靠近氨基端的PTB域是Numb作用的关键部位,可与E3泛素连接酶Mdm2,Lnx,Siahl,Itch相互作用(能够将目的蛋白质多聚泛素化,进而被蛋白酶体降解)。在Numb与肿瘤形成的研究中发现,Numb通过参与维持细胞极性和不对称分裂,上皮间质转化,蛋白质泛索化降解,抑制Notch, Hedgehog-Gli(简称Hh-Gli或Hh)信号传导通路,上调P53活性而广泛参与细胞增殖与分化、凋亡与再生等肿瘤发生的众多重要的病理生理过程,发挥其抑制肿瘤形成的作用。最近的研究表明,Numb在乳腺癌,非小细胞肺癌,唾液腺癌,成神神管细胞瘤等某些人类肿瘤中都有不同程度的表达缺关；在对人小脑髓母细胞瘤的研究中发现Numb能够通过介导Itch依赖的泛素化降解Glil,从而抑制Hh-Gli通路。然而,Numb在MPM中的表达、对Glil的调控作用以及Numb与肿瘤形成的关系尚未见报道。
为了探讨Numb在MPM发生、发展中的作用,进一步以Numb为靶点进行基因治疗提供理论依据,本论文从以下三个方面进行了研究：
一. Numb.Gli1在MPM组织中的表达及其临床意义：
二. Numb在MPM细胞株NCI-H2452中的表达及对Glil的调控作用；
三.过表达Numb对NCI-H2452细胞增殖、凋亡和化疗敏感性的影响。
实验方法
一.探讨Numb.Gli1在MPM组织中的表达及其临床意义
免疫组织化学法检测Numb.Gli1在6l例MPM组织及22例正常胸膜组织中的表达,卡方检验进行组间比较；Kruskal-Wall i s或Mann-Whitney法检测Numb、Gli1在MPM中表达与临床病理参数之间的关系；Spearman等级相关分析检验Numb与Gli1的相互关系；Kaplan-Meier法log-rank检验进行单因素生存分析,Cox回归进行多因素生存分析,检测Numb、Gli1及各项临床病理参数对预后的影响。
二.研究Numb在MPM细胞株NCI-H2452中的表达及对Glil的调控作用
1.首先用qRT-PCR及Western blot方法检测了Numb在NCI-H2452细胞中的表达,同时我们将已被证明表达Numb的人胚肾细胞293T作为对照；Western blot方法检测蛋白酶体抑制剂MG132作用于NCI-12452细胞后对Numb蛋白表达的影响。
2.Numb农达载体体外瞬时转染NCI-H2452细胞,Western blot方法检测Numb、Glil?达；蛋白酶体抑制剂MG132作用于Numb转染组,Western blot方法检测对Gli1表达的影响。
三.检测过表达Numb对MPM细胞株NCI-H2452增殖、凋亡和化疗敏感性的影响
1.为检测过表达Numb对NCI-H2452细胞增殖能力的影响,分别于转染后48、72h采用MTT法检测细胞存活率。
2.为检测过农达Numb对NCI-H2452细胞凋亡的影响,转染后72h采用Hoeehst染色和Annexin V-FITC/PI双染流式细胞术分别检测细胞凋亡形态的变化和凋亡率。
3.为研究Numb促进NCI-H2452细胞调亡的机制,采用Western blot方法检测凋亡相关蛋白caspase3,9以及细胞色素C、XIAP、survivin的变化。
4.为了进一步确定Numb对MPM化疗敏感性的影响,过表达Numb后分别加入3种不同的化疗药物顺铂、培美曲塞、卡铂作用24h,采用MTT方法检测细胞存活率。
结果
一. Numb. Gli1在MPM组织中的表达及其临床意义
Numb阳性表达率在MPM组明显低于正常对照组(P<0.05)；MPM组织中Numb表达与Ki-67标记指数密切相关,Ki-67<25%的患者中Numb表达强,Ki-67>25%的患者中Numb表达弱(P<0.05)。Glil阳性表达率在MPM组明显高于正常对照组(P<0.05)；Glil胞核表达与IMIG分期密切相关,IMIG分期为初期的患者Glil表达弱,进展期的患者Glil表达强(P<0.05)。在MPM组织中,Numb表达与Glil胞核表达呈显著负相关(r=-0.361,P<0.05)；Kaplan-Meier法log-rank检验进行单因素生存分析,结果显示在MPM组织中Numb表达缺失与预后不良有关(P<0.05)；此外,病理类型中的上皮型较之于非上皮型为有利预后因素(P<0.05)。Cox回归模型分析显示只有病理类型为独立的预后因素(HR=1.952,95%CI1.044,3.48P<0.05)。
二. Numb在MPM细胞株NCI-H2452中的表达及对Glil的调控作用
1. Numb在MPM细胞株NCI-H2452中的表达
结果显示NumbmRNA在NCI-H2452和293T细胞表达水平相似,但在蛋白水平,Numb在NCI-H2452细胞中的表达明显低于293T。经蛋白酶体抑制剂MG132作用后,Numb蛋白表达明显升高,提示Numb在NCI-H2452细胞中低表达或表达缺失与转录后抑制有关。
2.在NCI-H2452细胞中过表达Numb后Glil蛋白表达受到抑制
Numb蛋白在Numb转染组中表达明显高于空载体对照组,外源性表达Numb成功。
Gli1mRNA在Numb转染组和空载体对照组比较无明显差异：而Glil蛋白在Numb转染组较之于空载体对照组表达降低,提示Numb抑制了Glil蛋白表达。蛋白酶体抑制剂MG132作用于Numb转染组后,Gli1蛋白表达恢复,提示在MG132作用下Numb对Glil蛋白抑制作用消失。
三.过表达Numb对MPM细胞株NCI-H2452增殖、凋亡和化疗敏感性的影响
1.过表达Numb后NCI-H2452细胞增殖受到抑制
MTT检测结果显示转染外源性Numb基因48、72h后,Numb转染组与空载体对照组或空白对照组比较,OD值明显降低,结果有显著性差异(P<0.05,P<0.01)提示过表达Numb能够抑制NCI-H2452细胞增殖活力。
2.过表达Numb促进NCI-H2452细胞的凋亡
转染外源必忖Numb基天72h后,Hoechst染色在荧光显微镜下观察发现Numb转染组细胞凋亡显著,出现明显的核固缩,核碎裂,而空载体对照组和空白对照组凋亡细胞相对较少；Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率(早期凋亡率+晚期凋亡率)显示,Numb转染组与空载体对照组或空白对照组比较,凋亡率明显升高,结果有显著性差异(均P<0.01),提示过表达Numb舌,促进NCI-H2452细胞凋亡。
3. Numb通过激活caspase-9介导的内源性凋亡途径促进NCI-H2452细胞凋亡
Western blot结果显示同空载体对照组比较,Numb转染组细胞caspase-3,9活性蛋白和胞质细胞色索C表达明显升高,XIAP survivin表达减弱。提示Numb可能通过激活细胞色素C/caspase-9介导的内源性调亡途径促进NCI-H2452细胞凋亡。
4.过表达Numb增强NCI-H2452细胞对顺铂和卡铂的化疗敏感性。
MTT检测结果显示在相同浓度顺铂或卡铂作用下,Numb转染组的细胞存活率明显低于空载体对照组和空白对照组(P<0.05,P<0.01)；过表达Numb(?)培美曲塞作用的3组细胞存活率无明显影响。提示过表达Numb增强NCI-H2452细胞对铂类化疗药物的敏感性。
结论
1. Numb在MPM组织中的表达减弱甚至缺失,并且与高Ki-67标记指数及不良预后常切相关。
2.Glil在MPM组织中表达增强,成IMIG分期相关；Numb与Glil表达负相关,Numb(?)能通过泛索-蛋白酶体途径抑制Glil蛋白的表达。
3.过表达Numb能够抑制MPM细胞的增殖能力,并通过caspase-9介导的的内源性调亡途径促进MPM细胞的凋亡
4.过农达Numb能够增强MPM细胞对铂类化疗药物的敏感性。
创新性和意义
1.本课题首次发现Numb在MPM组织中表达减弱甚至缺失,并且为预后不良的指标。
2.本课题首次在MPM中研究了Numb与Glil的相关性及Numb对Glil的调控作用。
3.本课题首次在MPM中研究了Numb对肿瘤细胞增殖、凋亡及化疗敏感性的影响,为以Numb为靶点进行基因治疗提供了理论依据。
Background and Objective
Malignant pleural mesothelioma (MPM) is a rare and highly aggressive tumor with dismal prognosis. Although the asbestos exposure, which is regarded as the main risk for MPM, has been regulated since the1870s, the incidence is increasing continuedly because of the long latent period between exposure and clinical disease. Its mortality has been reported increasing over the past two decades in industrialized countries and is expected to peak in2010-2020. The histologic subtypes of mesothelioma include epithelioid (most common), sarcomatoid and mixed. MPM is poorly responsive to current oncological therapies with a median survival of around10-12months. In recent years, the success of gene therapy in tumor area lay a foundation for finding effective treatment for MPM.
Numb is one of the most important cell fate determinants and has been implicated as a tumor suppressor in organism. It is a membrane-associated protein which contains an amino-terminal phosphotyrosine-binding domain (PTB) and a C-terminal proline-rich site (PRR). The PTB domain is critical for Numb to interact with the E3-ligases Mdm2, Lnx, Siah1and Itch, which leads to poly-ubiquitination and proteolytic degradation of proteins. Numb has also been proven to participate in regulating the cell polarity and asymmetric cell division, epithelial-mesenchymal transition (KMT), ubiquitination and degradation, and down-regulating Notch and Hedgehog-Gli signaling while up-regulating TP53activity. Thus, Numb can inhibit the tumor formation through controlling the balance of proliferation and differentiation as well as apoptosis and regeneration of cells. Loss of Numb expression has been reported in some human cancers, such as breast cancers, non-small cell lung carcinomas, salivary gland carcinomas and medulloblastomas. A role for Numb in preventing tumorigenesis appears to involve the suppression of the oncogenic Hedgehog-Gli signaling. However, the expression and clinical significance of Numb in MPM and its effect on cancer development have not been reported.
To explore the effect of Numb in the development of MPM and lay a foundation for gene therapy, we investigated from the following three aspects:
1. The expression and clinical significance of Numb in MPM.
2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Gli1.
3. The effect of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells.
Methods
1. The expression of Numb and Gli1in MPM and their clinical significance
Numb and Gli1expression were evaluated by immunohistochemistry in61MPM tissues and22normal pleura, and Chi-square test was used to compare the results; Correlations of protein expression (grade scores) with clinical and pathological parameters of the tumors were evaluated with Kruskal-Wallis or Mann-Whitney test for ordinal variables. Correlations between Numb and Gli1expression were evaluated with the Spearman rank order correlation test. The univariate analysis of overall survival (OS) was carried out by the Kaplan-Meier method using the log-rank test. Multivariate Cox proportional hazards regression model was used to test for independency.
2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Glil
(1) First, the expression of Numb in NCI-H2452cells was detected by qRT-PCR and Western blot. Then we detected the protein expression of Numb in the presence of proteasome inhibitor MG132treatment.
(2) First, we transfected the pcDNA3.1Numb (Numb) or empty vector (mock) with LipofectaminTM2000into NCI-2452cells transiently and detected the protein expression of Numb and Glil by Western blot. Then we detected the protein expression of Gli1in Numb-transfected cells with the treatment of proteasome inhibitor MG132.
3.The effects of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells
(1) To explore the effect of Numb overexpression on the proliferation in NCI-H2452cells, MTT was used to detect survival rate at48h and72h after transfection.
(2) To explore the effect of Numb overexpression on the apoptosis in NCI-H2452cells, Hoechst33258staining and Annexin V-FITC/PI assay using flow cytometry were used to detect the change of cellular morphology and apoptosis rate at72h after transfection.
(3) To explore the mechanism of Numb overexpression enhancing apoptosis, the apoptosis related proteins caspase-3, caspase-9, cytochrome c, XIAP, survivin were detected by Western blot after transfection.
(4) To explore the effect of Numb overexpression on the sensibility to chemotherapy in NCI-H2452cells, MTT was used to detect survival rate with the treatment of cisplatin, pemetrexed or carboplatin at different concentrations.
Result
1.The expression of Numb and Glil in MPM and their clinical significance
The immunoreactivity of Numb in MPM was significantly lower than that of normal control group(P<0.05), and Numb has an inverse correlation with Ki-67labeling index (P<0.05). The immunoreactivity of Gli1in MPM was significantly higher than that of normal control group(P<0.05),and nuclear Gli1was found in associated with the tumor IMIG-stage (P<0.05).The expression levels of Numb with nuclear Glil exhibited a significant inverse correlation (r=-0.361P<0.05). Univariate analysis indicated that the OS was influenced by expression of Numb (P<0.05) and histological subtype (P<0.05), and further Cox regression analysis showed that only histological subtype was an independent prognostic factor for OS (P<0.05).
2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Gli1
(1) The expression of Numb in NCI-H2452cells
Before transfection, we detected comparable levels of NumbmRNA in NCI-112452vs. 293T cells, but the protein level of Numb in NCI-H2452was lower than that of293T cells. Numb protein was restored to a high level by treatment with the proteasome inhibitor MG132, which meaned that loss of Numb expression in NCI-H2452cells was determined at the post-translational level, through enhanced protein degradation.
(2) Numb overexpression in NCI-H2452cells inhibited Gli1protein expression The NCI-H2452cells transfected with pcDNA3.1Numb showed higher Numb protein expression compared with that of mock-transfected, While the Gli1protein expresssion was weakened. The Gli1protein in Numb-transfected cells restored to a high level with the treatment of proteasome inhibitor MG132, which meaned that Numb lost the inhibition effect on Glil in the presence of MG132.
3. The effects of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells
(1) Overexpression of Numb inhibited the proliferation in NCI-H2452cells The absorbance of Numb-transfected cells, as indicated by MTT, was markedly lower at48h and72h compared with that of mock-transfected cells or blank controls (P<0.05,<0.01), which meaned that Numb overexpression inhibited growth of NCI-H2452cells
(2) Overexpression of Numb promoted the apoptosis in NCI-H2452cells. Staining with Hoechst33258showed that Numb overexpression increased the number of apoptotic cells characterized by chromatin condensation and nuclear fragmentation, comparing with that of mock-transfected cells or blank controls; The apoptosis rate was determined by two-color analysis of Annexin V/FITC binding and propidium iodide (PI) uptake using flow cytometiy at72h after transfection.The percentages of early and late apoptotic cells ware notably higher in Numb-transfected than that of mock-transfected cells or blank controls (P<0.01).
(3) Numb induced apoptosis in NCI-H2452cells through the intrinsic caspase-9pathway We analyzed some proteins related to apoptosis in NCI-H2452cells by Western blot after transfection. Numb-transfected cells showed increased active caspase-9(35kDa) and caspase-3(17kDa) bands compared with that of mock-transfected cells. In addition, we found release of cytochrome c to cytoplasm as well as down-regulation of XIAP and survivin in Numb-transfected cells.
(4) Overexpression of Numb sensitized NCI-H2452cells to cisplatin and carboplatin The survival rate of Numb-transfected cells was significantly lower than that of mock-transfected cells and blank controls with the treatment of cisplatin and carboplatin at the same concentrations, which meaned that overexpression of Numb sensitized NCI-H2452cells to cisplatin and carboplatin.But the survival rates of the three groups had no statistically differences with the treatment of pemetrexed.
Conclusions
1. Numb expression was significantly decreased or complete absent in MPM tissues, which correlated with high Ki-67labeling index and poor prognosis.
2. Glil expression was significantly increased and correlated with IMIG stages in MPM tissues; Numb had an inverse correlation with Gli1and Numb overexpression inhibited Gli1protein through the ubiquitin-proteasome pathway.
3. Overexpression of Numb inhibited the proliferation and induced the apoptosis through the intrinsic caspase-9pathway in MPM cells.
4. Overexpression of Numb sensitized MPM cells to cisplatin and carboplatin.
Originality
1. We demonstrated, for the first time,that the frequent loss of Numb expression in MPM and it is a poor predictor of survival.
2. We investigated, for the first time, the correlation of Numb and Gli1as well as the regulation of Numb on Gli1.
3. We investigated, for the first time, the effect of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in MPM cells, which could lay a foundation for the gene therapy targeted Numb for MPM.
恶性胸膜间皮瘤(malignant pleural mesothelioma, MPM)是一种少见的胸膜部侵袭性肿瘤,预后极差。它的发病通常与接触石棉有关,尽管自19世纪70年代以来石棉的使用广受限制,但接触后的长期潜伏期使间皮瘤的发病率正持续上升。预计在2010-2020年间,工业发达国家恶性间皮瘤的死亡率将达到高峰。根据组织形态学分类,MPM可分为上皮型、肉瘤样型(纤维型)和混合型三种类型,以上皮型多见。本病传统治疗效果差,确诊后中位生存期约为10-12个月。因此,对MPM有效的诊疗措施亟待研究。近些年来,基因靶向治疗在肿瘤领域的成功应用为MPM寻求有效的治疗方法奠定了基础。
Numb是生物体内最重要的细胞命运决定因子之一,被认为参与多种恶性肿瘤的形成,被认为是颇具希望的抑癌基因。Numb是一种膜相关蛋白,其结构包含一个磷酸酪氨酸结合域(phosphotyrosine binding, PTB)和一个富脯氨酸域(proline rich region, PRR);靠近氨基端的PTB域是Numb作用的关键部位,可与E3泛素连接酶Mdm2,Lnx,Siahl,Itch相互作用(能够将目的蛋白质多聚泛素化,进而被蛋白酶体降解)。在Numb与肿瘤形成的研究中发现,Numb通过参与维持细胞极性和不对称分裂,上皮间质转化,蛋白质泛索化降解,抑制Notch, Hedgehog-Gli(简称Hh-Gli或Hh)信号传导通路,上调P53活性而广泛参与细胞增殖与分化、凋亡与再生等肿瘤发生的众多重要的病理生理过程,发挥其抑制肿瘤形成的作用。最近的研究表明,Numb在乳腺癌,非小细胞肺癌,唾液腺癌,成神神管细胞瘤等某些人类肿瘤中都有不同程度的表达缺关；在对人小脑髓母细胞瘤的研究中发现Numb能够通过介导Itch依赖的泛素化降解Glil,从而抑制Hh-Gli通路。然而,Numb在MPM中的表达、对Glil的调控作用以及Numb与肿瘤形成的关系尚未见报道。
为了探讨Numb在MPM发生、发展中的作用,进一步以Numb为靶点进行基因治疗提供理论依据,本论文从以下三个方面进行了研究：
一. Numb.Gli1在MPM组织中的表达及其临床意义：
二. Numb在MPM细胞株NCI-H2452中的表达及对Glil的调控作用；
三.过表达Numb对NCI-H2452细胞增殖、凋亡和化疗敏感性的影响。
实验方法
一.探讨Numb.Gli1在MPM组织中的表达及其临床意义
免疫组织化学法检测Numb.Gli1在6l例MPM组织及22例正常胸膜组织中的表达,卡方检验进行组间比较；Kruskal-Wall i s或Mann-Whitney法检测Numb、Gli1在MPM中表达与临床病理参数之间的关系；Spearman等级相关分析检验Numb与Gli1的相互关系；Kaplan-Meier法log-rank检验进行单因素生存分析,Cox回归进行多因素生存分析,检测Numb、Gli1及各项临床病理参数对预后的影响。
二.研究Numb在MPM细胞株NCI-H2452中的表达及对Glil的调控作用
1.首先用qRT-PCR及Western blot方法检测了Numb在NCI-H2452细胞中的表达,同时我们将已被证明表达Numb的人胚肾细胞293T作为对照；Western blot方法检测蛋白酶体抑制剂MG132作用于NCI-12452细胞后对Numb蛋白表达的影响。
2.Numb农达载体体外瞬时转染NCI-H2452细胞,Western blot方法检测Numb、Glil?达；蛋白酶体抑制剂MG132作用于Numb转染组,Western blot方法检测对Gli1表达的影响。
三.检测过表达Numb对MPM细胞株NCI-H2452增殖、凋亡和化疗敏感性的影响
1.为检测过表达Numb对NCI-H2452细胞增殖能力的影响,分别于转染后48、72h采用MTT法检测细胞存活率。
2.为检测过农达Numb对NCI-H2452细胞凋亡的影响,转染后72h采用Hoeehst染色和Annexin V-FITC/PI双染流式细胞术分别检测细胞凋亡形态的变化和凋亡率。
3.为研究Numb促进NCI-H2452细胞调亡的机制,采用Western blot方法检测凋亡相关蛋白caspase3,9以及细胞色素C、XIAP、survivin的变化。
4.为了进一步确定Numb对MPM化疗敏感性的影响,过表达Numb后分别加入3种不同的化疗药物顺铂、培美曲塞、卡铂作用24h,采用MTT方法检测细胞存活率。
结果
一. Numb. Gli1在MPM组织中的表达及其临床意义
Numb阳性表达率在MPM组明显低于正常对照组(P<0.05)；MPM组织中Numb表达与Ki-67标记指数密切相关,Ki-67<25%的患者中Numb表达强,Ki-67>25%的患者中Numb表达弱(P<0.05)。Glil阳性表达率在MPM组明显高于正常对照组(P<0.05)；Glil胞核表达与IMIG分期密切相关,IMIG分期为初期的患者Glil表达弱,进展期的患者Glil表达强(P<0.05)。在MPM组织中,Numb表达与Glil胞核表达呈显著负相关(r=-0.361,P<0.05)；Kaplan-Meier法log-rank检验进行单因素生存分析,结果显示在MPM组织中Numb表达缺失与预后不良有关(P<0.05)；此外,病理类型中的上皮型较之于非上皮型为有利预后因素(P<0.05)。Cox回归模型分析显示只有病理类型为独立的预后因素(HR=1.952,95%CI1.044,3.48P<0.05)。
二. Numb在MPM细胞株NCI-H2452中的表达及对Glil的调控作用
1. Numb在MPM细胞株NCI-H2452中的表达
结果显示NumbmRNA在NCI-H2452和293T细胞表达水平相似,但在蛋白水平,Numb在NCI-H2452细胞中的表达明显低于293T。经蛋白酶体抑制剂MG132作用后,Numb蛋白表达明显升高,提示Numb在NCI-H2452细胞中低表达或表达缺失与转录后抑制有关。
2.在NCI-H2452细胞中过表达Numb后Glil蛋白表达受到抑制
Numb蛋白在Numb转染组中表达明显高于空载体对照组,外源性表达Numb成功。
Gli1mRNA在Numb转染组和空载体对照组比较无明显差异：而Glil蛋白在Numb转染组较之于空载体对照组表达降低,提示Numb抑制了Glil蛋白表达。蛋白酶体抑制剂MG132作用于Numb转染组后,Gli1蛋白表达恢复,提示在MG132作用下Numb对Glil蛋白抑制作用消失。
三.过表达Numb对MPM细胞株NCI-H2452增殖、凋亡和化疗敏感性的影响
1.过表达Numb后NCI-H2452细胞增殖受到抑制
MTT检测结果显示转染外源性Numb基因48、72h后,Numb转染组与空载体对照组或空白对照组比较,OD值明显降低,结果有显著性差异(P<0.05,P<0.01)提示过表达Numb能够抑制NCI-H2452细胞增殖活力。
2.过表达Numb促进NCI-H2452细胞的凋亡
转染外源必忖Numb基天72h后,Hoechst染色在荧光显微镜下观察发现Numb转染组细胞凋亡显著,出现明显的核固缩,核碎裂,而空载体对照组和空白对照组凋亡细胞相对较少；Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率(早期凋亡率+晚期凋亡率)显示,Numb转染组与空载体对照组或空白对照组比较,凋亡率明显升高,结果有显著性差异(均P<0.01),提示过表达Numb舌,促进NCI-H2452细胞凋亡。
3. Numb通过激活caspase-9介导的内源性凋亡途径促进NCI-H2452细胞凋亡
Western blot结果显示同空载体对照组比较,Numb转染组细胞caspase-3,9活性蛋白和胞质细胞色索C表达明显升高,XIAP survivin表达减弱。提示Numb可能通过激活细胞色素C/caspase-9介导的内源性调亡途径促进NCI-H2452细胞凋亡。
4.过表达Numb增强NCI-H2452细胞对顺铂和卡铂的化疗敏感性。
MTT检测结果显示在相同浓度顺铂或卡铂作用下,Numb转染组的细胞存活率明显低于空载体对照组和空白对照组(P<0.05,P<0.01)；过表达Numb(?)培美曲塞作用的3组细胞存活率无明显影响。提示过表达Numb增强NCI-H2452细胞对铂类化疗药物的敏感性。
结论
1. Numb在MPM组织中的表达减弱甚至缺失,并且与高Ki-67标记指数及不良预后常切相关。
2.Glil在MPM组织中表达增强,成IMIG分期相关；Numb与Glil表达负相关,Numb(?)能通过泛索-蛋白酶体途径抑制Glil蛋白的表达。
3.过表达Numb能够抑制MPM细胞的增殖能力,并通过caspase-9介导的的内源性调亡途径促进MPM细胞的凋亡
4.过农达Numb能够增强MPM细胞对铂类化疗药物的敏感性。
创新性和意义
1.本课题首次发现Numb在MPM组织中表达减弱甚至缺失,并且为预后不良的指标。
2.本课题首次在MPM中研究了Numb与Glil的相关性及Numb对Glil的调控作用。
3.本课题首次在MPM中研究了Numb对肿瘤细胞增殖、凋亡及化疗敏感性的影响,为以Numb为靶点进行基因治疗提供了理论依据。
Background and Objective
Malignant pleural mesothelioma (MPM) is a rare and highly aggressive tumor with dismal prognosis. Although the asbestos exposure, which is regarded as the main risk for MPM, has been regulated since the1870s, the incidence is increasing continuedly because of the long latent period between exposure and clinical disease. Its mortality has been reported increasing over the past two decades in industrialized countries and is expected to peak in2010-2020. The histologic subtypes of mesothelioma include epithelioid (most common), sarcomatoid and mixed. MPM is poorly responsive to current oncological therapies with a median survival of around10-12months. In recent years, the success of gene therapy in tumor area lay a foundation for finding effective treatment for MPM.
Numb is one of the most important cell fate determinants and has been implicated as a tumor suppressor in organism. It is a membrane-associated protein which contains an amino-terminal phosphotyrosine-binding domain (PTB) and a C-terminal proline-rich site (PRR). The PTB domain is critical for Numb to interact with the E3-ligases Mdm2, Lnx, Siah1and Itch, which leads to poly-ubiquitination and proteolytic degradation of proteins. Numb has also been proven to participate in regulating the cell polarity and asymmetric cell division, epithelial-mesenchymal transition (KMT), ubiquitination and degradation, and down-regulating Notch and Hedgehog-Gli signaling while up-regulating TP53activity. Thus, Numb can inhibit the tumor formation through controlling the balance of proliferation and differentiation as well as apoptosis and regeneration of cells. Loss of Numb expression has been reported in some human cancers, such as breast cancers, non-small cell lung carcinomas, salivary gland carcinomas and medulloblastomas. A role for Numb in preventing tumorigenesis appears to involve the suppression of the oncogenic Hedgehog-Gli signaling. However, the expression and clinical significance of Numb in MPM and its effect on cancer development have not been reported.
To explore the effect of Numb in the development of MPM and lay a foundation for gene therapy, we investigated from the following three aspects:
1. The expression and clinical significance of Numb in MPM.
2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Gli1.
3. The effect of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells.
Methods
1. The expression of Numb and Gli1in MPM and their clinical significance
Numb and Gli1expression were evaluated by immunohistochemistry in61MPM tissues and22normal pleura, and Chi-square test was used to compare the results; Correlations of protein expression (grade scores) with clinical and pathological parameters of the tumors were evaluated with Kruskal-Wallis or Mann-Whitney test for ordinal variables. Correlations between Numb and Gli1expression were evaluated with the Spearman rank order correlation test. The univariate analysis of overall survival (OS) was carried out by the Kaplan-Meier method using the log-rank test. Multivariate Cox proportional hazards regression model was used to test for independency.
2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Glil
(1) First, the expression of Numb in NCI-H2452cells was detected by qRT-PCR and Western blot. Then we detected the protein expression of Numb in the presence of proteasome inhibitor MG132treatment.
(2) First, we transfected the pcDNA3.1Numb (Numb) or empty vector (mock) with LipofectaminTM2000into NCI-2452cells transiently and detected the protein expression of Numb and Glil by Western blot. Then we detected the protein expression of Gli1in Numb-transfected cells with the treatment of proteasome inhibitor MG132.
3.The effects of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells
(1) To explore the effect of Numb overexpression on the proliferation in NCI-H2452cells, MTT was used to detect survival rate at48h and72h after transfection.
(2) To explore the effect of Numb overexpression on the apoptosis in NCI-H2452cells, Hoechst33258staining and Annexin V-FITC/PI assay using flow cytometry were used to detect the change of cellular morphology and apoptosis rate at72h after transfection.
(3) To explore the mechanism of Numb overexpression enhancing apoptosis, the apoptosis related proteins caspase-3, caspase-9, cytochrome c, XIAP, survivin were detected by Western blot after transfection.
(4) To explore the effect of Numb overexpression on the sensibility to chemotherapy in NCI-H2452cells, MTT was used to detect survival rate with the treatment of cisplatin, pemetrexed or carboplatin at different concentrations.
Result
1.The expression of Numb and Glil in MPM and their clinical significance
The immunoreactivity of Numb in MPM was significantly lower than that of normal control group(P<0.05), and Numb has an inverse correlation with Ki-67labeling index (P<0.05). The immunoreactivity of Gli1in MPM was significantly higher than that of normal control group(P<0.05),and nuclear Gli1was found in associated with the tumor IMIG-stage (P<0.05).The expression levels of Numb with nuclear Glil exhibited a significant inverse correlation (r=-0.361P<0.05). Univariate analysis indicated that the OS was influenced by expression of Numb (P<0.05) and histological subtype (P<0.05), and further Cox regression analysis showed that only histological subtype was an independent prognostic factor for OS (P<0.05).
2. The expression of Numb in MPM cell line NCI-H2452and its regulation on Gli1
(1) The expression of Numb in NCI-H2452cells
Before transfection, we detected comparable levels of NumbmRNA in NCI-112452vs. 293T cells, but the protein level of Numb in NCI-H2452was lower than that of293T cells. Numb protein was restored to a high level by treatment with the proteasome inhibitor MG132, which meaned that loss of Numb expression in NCI-H2452cells was determined at the post-translational level, through enhanced protein degradation.
(2) Numb overexpression in NCI-H2452cells inhibited Gli1protein expression The NCI-H2452cells transfected with pcDNA3.1Numb showed higher Numb protein expression compared with that of mock-transfected, While the Gli1protein expresssion was weakened. The Gli1protein in Numb-transfected cells restored to a high level with the treatment of proteasome inhibitor MG132, which meaned that Numb lost the inhibition effect on Glil in the presence of MG132.
3. The effects of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in NCI-H2452cells
(1) Overexpression of Numb inhibited the proliferation in NCI-H2452cells The absorbance of Numb-transfected cells, as indicated by MTT, was markedly lower at48h and72h compared with that of mock-transfected cells or blank controls (P<0.05,<0.01), which meaned that Numb overexpression inhibited growth of NCI-H2452cells
(2) Overexpression of Numb promoted the apoptosis in NCI-H2452cells. Staining with Hoechst33258showed that Numb overexpression increased the number of apoptotic cells characterized by chromatin condensation and nuclear fragmentation, comparing with that of mock-transfected cells or blank controls; The apoptosis rate was determined by two-color analysis of Annexin V/FITC binding and propidium iodide (PI) uptake using flow cytometiy at72h after transfection.The percentages of early and late apoptotic cells ware notably higher in Numb-transfected than that of mock-transfected cells or blank controls (P<0.01).
(3) Numb induced apoptosis in NCI-H2452cells through the intrinsic caspase-9pathway We analyzed some proteins related to apoptosis in NCI-H2452cells by Western blot after transfection. Numb-transfected cells showed increased active caspase-9(35kDa) and caspase-3(17kDa) bands compared with that of mock-transfected cells. In addition, we found release of cytochrome c to cytoplasm as well as down-regulation of XIAP and survivin in Numb-transfected cells.
(4) Overexpression of Numb sensitized NCI-H2452cells to cisplatin and carboplatin The survival rate of Numb-transfected cells was significantly lower than that of mock-transfected cells and blank controls with the treatment of cisplatin and carboplatin at the same concentrations, which meaned that overexpression of Numb sensitized NCI-H2452cells to cisplatin and carboplatin.But the survival rates of the three groups had no statistically differences with the treatment of pemetrexed.
Conclusions
1. Numb expression was significantly decreased or complete absent in MPM tissues, which correlated with high Ki-67labeling index and poor prognosis.
2. Glil expression was significantly increased and correlated with IMIG stages in MPM tissues; Numb had an inverse correlation with Gli1and Numb overexpression inhibited Gli1protein through the ubiquitin-proteasome pathway.
3. Overexpression of Numb inhibited the proliferation and induced the apoptosis through the intrinsic caspase-9pathway in MPM cells.
4. Overexpression of Numb sensitized MPM cells to cisplatin and carboplatin.
Originality
1. We demonstrated, for the first time,that the frequent loss of Numb expression in MPM and it is a poor predictor of survival.
2. We investigated, for the first time, the correlation of Numb and Gli1as well as the regulation of Numb on Gli1.
3. We investigated, for the first time, the effect of Numb overexpression on the proliferation, apoptosis and chemotherapy sensitivity in MPM cells, which could lay a foundation for the gene therapy targeted Numb for MPM.
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