【作者】 徐若冰；

【导师】 张雅洁；

【作者基本信息】 南方医科大学 ， 病理与病理生理学， 2008， 博士

【摘要】 研究背景:肺癌是目前世界上发病率和死亡率最高的恶性肿瘤,其中非小细胞肺癌(Non-small cell lung cancer,NSCLC)约占80%,侵袭和转移是其主要的致死原因,因而研究NSCLC侵袭转移的分子机制、寻找相关的分子标记和靶点就成为当前迫切需要解决的问题。肿瘤转移是多阶段、多步骤、多基因参与的过程,涉及多种癌基因的激活和抑癌基因的失活,其中抑癌基因的异常在肿瘤转移中的作用越来越受到重视。新的抑癌基因DAL-1(Differentially expressed in Adenocarcinoma of theLung)是美国科学家Tran YK于1999年通过差异显示PCR技术(DDPCR)发现在肺腺癌中表达缺失而正常肺组织有表达的一个基因,其染色体定位于18p11.3,编码蛋白属膜相关蛋白家族4.1亚类,为细胞骨架蛋白。免疫细胞化学研究显示DAL-1定位于细胞膜,呈“honeycomb”状分布,参与细胞-细胞间的连接和细胞黏附,由此推测该基因表达缺失有可能削弱细胞间的连接和黏附而导致肿瘤细胞易发生脱落,可能是肿瘤转移的重要分子基础。目前研究发现,在肺癌、乳腺癌、脑膜瘤等都存在染色体18p11.3区域的杂合缺失,在NSCLC中DAL-1基因表达缺失率超过50%。研究证明DAL-1基因表达缺失与脑膜瘤发生有关,是肿瘤发生的早期事件。但DAL-1基因与肺癌转移的关系尚未见报道。因而研究DAL-1与NSCLC转移的关系,对于阐明其转移机制及寻找新的肿瘤转移治疗靶点具有重要意义。RNA干扰(RNAi,RNA interference)是指某些小的双链RNA(Smallinterfering RNA,siRNA)可以特异、高效地与靶mRNA序列结合,导致mRNA降解,诱导细胞转化为特定基因低表达或缺失的表型。RNAi作为一种简单有效的替代基因敲除的工具,目前已广泛应用于功能基因组学研究和基因治疗等领域。蛋白质组学是后基因组时代计划中最重要的组成部分,它使人们从组织或细胞蛋白质的整体水平研究肿瘤成为可能。应用比较蛋白质组学技术可以发现肿瘤相关的特异性蛋白质。将RNA干扰与差异蛋白质组学相结合,为研究单基因敲除导致的细胞蛋白质谱改变提供了非常便捷的途径,为揭示肿瘤发生的分子机制、肿瘤诊断、防治、预后评估及新药开发等提供了新的研究方法。抑癌基因的失活可以由遗传学和表观遗传学机制导致。近来的研究已明确,表观遗传学的改变在某些肿瘤抑制基因或生长负调控基因的失活过程中作用尤为突出,其中启动子CpG岛甲基化是造成肿瘤抑制基因功能失活的重要表观遗传学机制之一。因为甲基化只引起基因表达异常,并不改变DNA本身的序列和基因产物。因此,运用甲基化抑制剂使已发生甲基化的基因去甲基化已成为基因功能研究的常用方法,也可能成为一种肿瘤治疗的新途径。研究目的:本研究重点探讨DAL-1基因在NSCLC细胞迁移、侵袭中的作用,并初步探讨其可能的作用机制,该基因在NSCLC中主要的失活原因及其可能的治疗途径,以期为NSCLC的临床治疗寻找新的靶点及提供实验理论依据。研究方法:1.选择临床确证伴有远处转移的NSCLC病人胸水分离培养细胞系和NSCLC细胞株为研究对象,用RT-PCR和Western blot方法检测DAL-1mRNA和蛋白表达情况,分析DAL-1基因表达与NSCLC侵袭的关系。2.根据人DAL-1mRNA编码序列,设计并合成靶向DAL-1基因的4条特异性DAL-1shRNA表达质粒pGPU6/GFP/Neo一T1、T2、T3、T4和阴性对照质粒(TN,pGPU6/GFP/Neo-TN);在阳离子脂质体介导下,用重组质粒转染NCI-H460细胞株(在①中检测DAL-1高表达),筛选出稳定转染细胞进行克隆,采用RT-PCR和Western blot方法检测DAL-1基因表达情况,分别计算表达抑制率。用MTT法检测DAL-1基因沉默后细胞增殖活性,Transwell运动小室实验检测细胞迁移和侵袭能力,探讨DAL-1对NSCLC细胞侵袭能力的影响。3.RNAi结合差异蛋白质组学方法,筛选出DAL-1沉默前后的差异蛋白并鉴定之,通过DAL-1作用相关蛋白的分析,探讨DAL-1对NSCLC细胞侵袭能力的影响及可能的作用机制。4.应用MSP法检测DAL-1表达缺失的细胞株和细胞系中DAL-1启动子的甲基化情况,探讨DAL-1基因失活与其启动子甲基化之间的关系。5.用5-aza-dc处理已发生DAL-1启动子甲基化的A549细胞,MSP法检测5-aza-dc的去甲基化效能;用RT-PCR和western blot方法检测处理后DAL-1mRNA和蛋白表达情况;采用Transwell实验检测恢复DAL-1表达后细胞的运动和侵袭能力的改变,分析5-aza-dc处理是否能恢复DAL-1的表达和功能。研究结果:1.DAL-1基因在NSCLC细胞株和细胞系中的表达在10个伴有远隔转移的NSCLC患者胸水分离培养的细胞系中,有9个DAL-1表达呈阴性,仅有1例(L8)为阳性;7个NSCLC细胞株中,有5个DAL-1表达呈阴性,2个为阳性(NCI-H460、GLC-82)。DAL-1在mRNA和蛋白水平表达一致。2.RNAi沉默DAL-1基因在NCI-H460细胞的作用自行设计构建靶向DAL-1的pGPU6/GFP/Neo—DAL-1shRNA质粒和阴性对照质粒pGPU6/GFP/Neo-TN,建立了DAL-1稳定沉默的NCI-H460/T和阴性对照NCI-H460/TN细胞株。与阴性和空质粒对照比较,荧光定量PCR方法检测T4转染组的NCI-H460细胞DAL-1抑制率达87.40±1.994%,Western blot结果显示T4转染组的NCI-H460细胞DAL-1抑制率达到(82.72±2.147)%,认为该组可以有效抑制DAL-1表达,命名为NCI-H460/T。NCI-H460/TN与空质粒转染组NCI-H460/ET抑制率没有显著性差异。3.DAL-1基因沉默对NCI-H460细胞生物学行为的影响MTT法检测NCI-H460/T细胞与NCI-H460/TN的增殖情况,与阴性对照组NCI-H460/TN和空质粒转染组NCI-H460/ET相比,NCI-H460/T增殖速度加快,组间有显著性差异(P=0.000)。Transwell迁移小室实验检测NCI-H460/T细胞的迁移能力,与阴性对照组NCI-H460/TN和空质粒转染组NCI-H460/ET比较,迁移能力均明显增强,统计学分析显示有显著性差异(P=0.000)。Transwell迁移小室实验检测NCI-H460/T细胞的侵袭能力,与阴性对照组NCI-H460/TN和空质粒转染组NCI-H460/ET比较,侵袭能力均明显增强,统计学分析显示有显著性差异(P=0.000)。4.DAL-1作用相关蛋白的分析对DAL-1沉默的NCI-H460/T细胞和阴性对照NCI-H460/TN细胞,通过双向电泳,以永生化的人支气管上皮细胞为参照,共筛选出10个表达差异达5倍以上的蛋白质点,进行质谱鉴定。在NCI-H460/T中有5个蛋白表达上调,其中Stathmin、Annexin I的上调与细胞增殖能力正相关,CCT2、HNRPH1、Moesin的上调则与细胞的侵袭、高转移能力相关;4个蛋白表达下调或不表达,其中peroxiredoxinⅠ、Ⅲ和Mn-SOD的下调以及peroxiredoxinⅡ的不表达与细胞恶性转化、退分化及细胞的高增殖、高侵袭能力相关;GST-Pi的下调机制不明,有待进一步研究。5.DAL-1表达缺失的NSCLC细胞系和细胞株中DAL-1启动子的甲基化检测采用MSP方法检测了DAL-1阴性表达的9个细胞系和5个细胞株,其中有6个细胞系和2个细胞株启动子有甲基化。但尚有3个细胞系和3个细胞株中DAL-1表达缺失,但其启动子未检出甲基化,表明尚有其它原因可导致DAL-1失活。6.5-aza-dc对DAL-1启动子甲基化的A549细胞株去甲基化处理选择有DAL-1启动子甲基化的A549细胞株,用5-aza-dc处理后可以有效逆转其甲基化,RT-PCR和Western blot检测可以恢复A549细胞的DAL-1表达。Transwell迁移和侵袭小室实验检测恢复DAL-1表达后A549细胞的运动和侵袭能力显著减弱,与对照组之间具有显著性差异(P=0.000)。结论:1.DAL-1基因表达缺失是NSCLC细胞株和细胞系中的常见事件,可能与其转移有关;DAL-1基因表达缺失不是由转录后机制所致。2.成功构建了靶向DAL-1的pGPU6/GFP/Neo-DAL-1shRNA质粒和阴性对照质粒pGPU6/GFP/Neo-TN;建立了DAL-1稳定沉默的NCI-H460/T和阴性对照NCI-H460/TN细胞株。沉默DAL-1基因后,NCI-H460细胞增殖、迁移和侵袭能力增强,提示DAL-1能有效抑制NSCLC细胞的增殖、迁移和侵袭。3.采用RNA干扰结合差异蛋白质组学技术对NCI-H460/T和NCI-H460/TN进行对比检测,筛选出10个差异蛋白质,经鉴定、分析,其中9个蛋白质的作用与细胞增殖、迁移和侵袭有关。通过对DAL-1作用相关蛋白的分析,也提示DAL-1有抑制NSCLC细胞增殖、迁移和侵袭的作用。详细作用机制有待进一步研究。4.启动子甲基化是DAL-1基因失活的重要原因之一。5.5-aza-dc可有效逆转A549细胞DAL-1基因启动子的甲基化状态,恢复DAL-1基因的表达和功能。综上所述,DAL-1基因与NSCLC侵袭密切相关,其抑制侵袭的作用部分是通过调节转移相关蛋白表达实现的;启动子甲基化是DAL-1基因失活的重要原因之一;去甲基化处理可有效恢复DAL-1基因的表达及功能,因而,去甲基化可能成为NSCLC临床治疗的一个新途径。本研究创新性之处1、自行设计靶向DAL-1的siRNA序列,构建pGPU6/GFP/Neo-DAL-1shRNA表达载体,研究证明可有效抑制NCI-H460细胞的DAL-1表达,建立DAL-1稳定沉默的NCI-H460细胞株,为今后研究DAL-1在肺癌中的作用机制提供了理想的细胞模型;2、首次选用临床确证伴有远处转移的NSCLC病人胸水分离细胞系作为研究对象,研究DAL-1与NSCLC侵袭转移的关系;3、采用RNAi结合差异蛋白质组学技术从相关蛋白质作用角度分析DAL-1在NCI-H460细胞增殖、迁移和侵袭的作用,从其作用相关蛋白网络的角度分析DAL-1与NSCLC转移的关系并初步探讨其作用机制,为进一步研究非小细胞肺癌发生、发展提供实验基础。更多还原

【Abstract】 Background:Lung cancer is one of the most high-incidence malignancies in the world at present.About 80%of lung cancers are non-small cell lung cancers(NSCLC).The main cause of death in NSCLC patients is the invasion and metastasis of tumor.So it becomes the present big issue to investigate the molecular mechanism of NSCLC invasion and metastasis and to look for novel related molecular marker and target. The metastasis of tumor is a complicated process,lots of stages,steps and genes are involved.Some oncogenes are activated and some tumor suppressor genes are deactivated.It becomes more and more important that the effects of the dysfunction of tumor suppressor gene in metastasis of tumor.DAL-1(Differentially expressed in Adenocarcinoma of the Lung) is a novel tumor suppressor gene which was found negatively expressed in adenocarcinoma as positively expressed in normal tissue of the lung by Tran YK,et al,by DDPCR assay in 1999.This gene is located in chromosome 18p11.3,encoding a cyto-skeleton protein belonged to membrane-related protein 4.1 family.The location of DAL-1 is in cell membrane by cyto-immunochemistry assay,distributed as "honeycomb".DAL-1 is involved in cell connection and adhension.We suppose that the dysfunction of DAL-1 may weaken the cell-cell connection and adhension,so it becomes easier for tumor cell to emigrate. Perhaps this could be important molecular basis of tumor metastasis.It has been proved the loss of heterozygosis(LOH) of 18p11.3 and loss of 4.1B/DAL-1 in lung cancer,breast cancer and meningioma.Because of happening at early stage,the negatively expression of DAL-1 is related to the genesis of meningioma.The loss rate of DAL-1 expression is more than 80%in NSCLC,but there are few reports about the relationship of DAL-1 and NSCLC metastasis.So,for elucidation of the mechanism of NSCLC metastasis,novel tumor suppressor,and therapy target,it is so important to investigate the expression pattern,the function in procession of invasion, and the deactivation reason of 4.1B/DAL-1,one of the cytoskeleton protein which has complex function in NSCLC.RNA interference(RNAi) is a kind of reliable gene silencing techniques,which involves double-stranded RNA-mediated,sequence-specific and highly-efficient mRNA degradation.It could induce specific single strand mRNA degradation post-transcriptionally.RNA interference technologies are current techniques widely utilized in functional genomic and gene-therapy studies.As the human genome project(HGP) has finished,the investigation of protein, which is the production of gene and directly functional representative of cell,gets more and more hot,and the proteomics,as the most important part of post-genomics project,is widely investigated also.By proteomics technology,it is possible to investigate tumor at global protein level.Special tumor-related proteins can be found by comparative proteomic technology.To combind RNAi and the comparative proteomic technology Applied a convenient methods to research on cyto-proteomics changes caused by mono gene knock-out,It is possible to find special tumor-related proteins to reveal the molecular mechanism of tumor genesis,diagnosis,prevention and cure,evaluation of prognosis,and new drug development of tumor by using comparative proteomic technology.Dysfunction of tumor suppressor gene can be caused by genetic and Epigenentic mechanisms.Recent researches showed that epigenetic variations,especially i methylation of CpG islands in promoter are very important in Dysfunction of tumor suppressor gene.The modification of methylation only causes the abnormality of gene expression,no effects on the sequence or products of DNA.So to demethylate the gene which promoter is methylated by methylation inhibitor becomes a general method of gene function study,it is also possible to become a new way of tumor therapyObjects:This study emphasized on the effects of DAL-1 gene in processions of cell emigration and invasion of NSCLC cell,the possible mechanism.,the reasons of deactivation of DAL-1 in NSCLC and possible therapy.We wish that our research could offer some experimental proves of the novel target and new therapy for clinical treatment of NSCLC.Methods:1.We chose the cultured cell lines separated from pleural fluid of NSCLC patients with distant metastasis(bone or brain) clinically diagnosed and NSCLC cell strains to detect the DAL-1 expression and the level of deactivation by RT-PCR and western blot,so we can analyze the relationship between the expression of DAL-1 and metastasis of NSCLC.2.According to the sequence of DAL-1 mRNA,we design and construct 4 specific shRNA interference plasmid vectors targeted to DAL-1 gene mRNA and 1 negative control plasmid vector were designed and synthesized.The pGPU6/GFP/Neo plasmid was used to constructed the specific RNA interference vector(pGPU6/GFP/Neo-T1,T2,T3,T4) aimed at DAL-1 mRNA and pGPU6/GFP/Neo-TN contained a free correlation sequence.The constructed shRNA expression vectors of positive clones were validated by double-enzyme digestion and DNA sequencing;Co-transfect NCI-H460 cell(DAL-1 positive chosen in Chap.1) with pGPU6/GFP/Neo-T1,T2,T3,T4,pGPU6/GFP/Neo-TN and pGPU6/GFP/Neo by cationic lipsome.Screen positive stable transfected cell clones.Detect the change of DAL-1 mRNA with RT-PCR and DAL-1 protein expression with Western blot.The change of Cell proliferation activity by MTT assay,measure capability change of emigration and invasion in DAL-1 silenced NCI-H460 cell.Analyze the relationship between DAL-1 and NSCLC invasion.DAL-1 in NCI-H460 cell strain was silenced by RNA interference technology, the invasion-promoting effects were observed and the possible mechanisms were investigated by proteomic techniques;to A549 which DAL-1 promoter is methylated, we use 5-aza-dc to treat cell,and detect the DAL-1 expression after treatment,the changes of cell amplification,migration and invasion capabilities were also observed3.We used the method combined RNAi and comparative proteomic technology to screen the differentially expressed protein,investigate the effects and possible mechanism of DAL-1 in NSCLC invasion through analyzing the DAL-1 function-related proteins.4.In cell lines and strains in which DAL-1 was negatively expressed,we use methylation-specific PCR(MSP) to detect the methylation of DAL-1 promoter,the relationship between DAL-1 deactivation and the methylation of DAL-1 promoter were studied;5.We use 5-aza-dc to treat A549 which DAL-1 promoter is methylated,detect the unmethylation efficiency of 5-aza-dc by MSP assay,detect the DAL-1 mRNA and protein expression after treatment,the changes of cell migration and invasion capability were also observed.The relationship between the treatment of 5-aza-dc and reactivation of DAL-1 expression and function was studied.Main results:1.The DAL-1 expression in NSCLC cell lines and strains9 of 10 NSCLC cell lines which were separated from pleura fluid of clinical diagnosed patients with distant metastasis(bone or brain) have no expression of DAL-1 at both mRNA and protein level,only 1 in 10(L8) is DAL-1 positive;5 of 7 NSCLC cell strains neither,the other 2 are positive(NCI-H460 and GLC-82).The expression of DAL-1 is same at both mRNA and protein level indicates that the loss of DAL-1 is not caused by post-transcription mechanisms2.The effects of DAL-1 silencing by RNAi assay in NCI-H460 cellWe used pGPU6/GFP/Neo plasmid to construct the specific RNA interference vector,pGPU6/GFP/Neo-DAL-lshRNA(T4) aimed at DAL-lgene mRNA and pGPU6/GFP/Neo-TN which contains a free correlation sequence;The mRNA expression level of DAL-1 was detected by real-time quantative RT-PCR,the mRNA expression level of DAL-1 of T4 group decreased(87.40±1.994)% respectively of that in NCI-H460/TN;Western blot analysis showed the DAL-1 protein decreased(82.72±2.147)%of T4 group respectively of that in NCI-H460/TN as well.T4 group was the cell line which DAL-1 gene was knockdown efficiently and named NCI-H460/T.The expression of DAL-1 at both mRNA and protein level has no difference between NCI-H460/TN and NCI-H460/ET (Empty plasmid Transfection).So we established NCI-H460/T cell line which had successful and stable inhibition of DAL-1 expression and NCI-H460/TN cell line which acted as a negative control.3.The effects of DAL-1 silencing on cellular biological behavior of NCI-H460MTT assay showed that NCI-H460/T cell proliferation was significantly enhanced compared with that in NCI-H460/TN(P=0.000),but there was no significant difference between NCI-H460/TN and NCI-H460/ET.The capabilities of migration and invasion of NCI-H460/T cell significantly enhanced detected by the mobility assay and the matrigel-invasive mobility assay,statistical analysis shows significant difference(P=0.000).4.Analysis of DAL-1 function-related proteinsWe compared the proteomic changes between NCI-H460/T and NCI-H460/TN by 2-dimensional gel electrophoresis(2-DE),As normal human bronchi epithelial cell strain(HBE) is used for reference,10 differentially expressed proteins between the 2 cell lines were identified by MALDI-TOF-MS(matrix-assisted laser desorption/ionization mass spectrometry).,Among 10 differentially expressed protein, 5 of them including were up-regulated in NCI-H460/T,Stathmin and annexin I are related to high-proliferation of tumor cell,CCT2,HNRPH1and moesin are related to aggressive capability of invasion and metastasis.While 4 of them including peroxiredoxinⅠ、Ⅲ、Mn-SOD were down-regulated in NCI-H460/T,and 1 of them, which is peroxiredoxinⅡwas negatively expressed in NCI-H460/T.These changes are related to malignant transforming,dedifferenciation,high proliferation and invasion.The mechanism of down-regulation of GST-Pi in NCI-H460/T is still unknown.5.The detection of promoter methylation of DAL-1 in cell lines and strains which DAL-1 are negative expressed.DAL-1 gene promoter methylation in 9 cell lines and 5 cell strains which DAL-1 are negatively expressed was detected by methylation-specific PCR(MSP).The results showed:that promoter methylation of DAL-1 gene was found in 6 of 9 cell lines and 2 of 5 cell strains.Promoter methylation of DAL-1 may be one of important reasons of the negative expression of DAL-1 protein and mRNA.However,no methylation was detected in 3 cell lines and 3 cell strains in which DAL-1 are negatively expressed indicates that loss of DAL-1 can be caused by other reasons.6.The unmethylation treatment of 5-aza-dc on A549 cell which DAL-1 promoter is methylated.A549 cell which DAL-1 promoter is methylated were treated with 5-aza-dc.The restoration of DAL-1 gene protein expression and mRNA levels were identified after treatment with 5-aza-dc.DAL-1 promoter methylation could not be detected any more. These results suggest that 5-aza-dc can successfully reverse the status of DAL-1 gene methylation and restore DAL-lgene expression.The capability of migration and invasion of A549 cells were decreased significantly(p=0.000) after treatment with 5-aza-dc by Transwell assay.Summary:1.The loss rate of DAL-1 is relatively high in NSCLC cell lines and strains.The loss of DAL-1 may relate to metastasis of NSCLC.The loss of DAL-1 is not cause by post-transcriptional mechanisms.2.We successfully constructed pGPU6/GFP/Neo-DAL-1-shRNA and pGPU6/GFP/Neo-TN plasmid vectors,constructed NCI-H460/T cell line in which DAL-1 is stable silenced and NCI-H460/TN as a negative control.Silencing of DAL-1 gene can enhance the proliferation,migration and invasion of NCI-H460 in vitro,the DAL-1 expression could inhibit the proliferation,migration and invasion of NSCLC.3.10 differentially expressed proteins related to DAL-1 were found by comparative proteomic study of NCI-H460/T and NCI-H460/TN.9/10 proteins are related to proliferation,migration and invasion of tumor cell.The results indicate that DAL-1 can inhibit proliferation,migration and invasion through analysis of DAL-1 function related proteins.The mechanism in detail would be studied in future.4.Methylation of DAL-1 promoter is one of the important reasons for DAL-1 loss.5.5-aza-dc can reverse the methylation of DAL-1 promoter and restore expression and function of DAL-1.As a tumor suppressor gene,DAL-1 has very important effects in invasion of NSCLC.DAL-1 can regulate its function-related proteins to inhibit invasion. Methylation of promoter is one of important reasons of DAL-1 dysfunction. Demethylation treatment can restore the expression and function of DAL-1,so demethylation can be a novel method for clinical therapy of NSCLC.更多还原