1.以基因组DNA改变鉴别非浸润性与浸润性膀胱癌的研究 2.HSPC300异常表达对非小细胞肺癌转移潜能影响的研究
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摘要
第一章:以基因组DNA改变鉴别非浸润性与浸润性膀胱癌的研究
手术前准确区分非浸润性或浸润性膀胱尿路上皮癌对于制定合理的临床治疗方案至关重要;然而,目前尚缺乏有效易行的鉴别诊断手段。本课题在建立稳定的微阵列比较基因组杂交(array comparative genome hybridization,array CGH)技术的基础上研究膀胱癌基因组DNA的异常,以期获得膀胱癌基因组DNA拷贝数改变谱型;进而揭示非浸润性与浸润性膀胱癌的特征性改变的基因或区域,并以这些DNA分子组合来区分两种类型的膀胱癌。这项研究的远期目标为,利用一组DNA分子标志物对非浸润性和浸润性膀胱癌进行手术前的辅助鉴别诊断,以指导正确治疗。
首先,应用Agilent Human Genome CGH 44B芯片,针对35例膀胱尿路上皮癌(其中非浸润性膀胱癌18例,浸润性膀胱癌17例)的组织样品基因组DNA进行了array CGH检测。继而,采用CGH Analytics 3.4(Agilent)和R project(Bioconductor)软件分析了35张芯片的实验数据。结果显示,除性染色体之外,膀胱癌组织基因组DNA拷贝数异常变化主要发生在1q、2q、3p、4q、5p、5q、7p、8p、8q、9p、9q、10q、11p、11q、13q、15q、16p、16q、17p、17q、18q等染色体臂,其中9号染色体改变的发生频率最高。而超过20%的高水平扩增区或纯合性缺失区分别为14个和11个。浸润性膀胱癌基因组DNA在5、8、10、17、18号染色体的改变显著高于非浸润性膀胱癌。经过对35例array CGH实验数据的生物信息学分析,初步筛选出8个能区分非浸润性与浸润性膀胱癌的基因(或区域)。然后以实时定量PCR技术在上述35例起始基因组DNA样品中进行验证,从而确认了array CGH分析结果的可靠性。
上述研究资料表明,在本实验室内已经建立起稳定可靠的array CGH分析技术;非浸润性与浸润性膀胱癌基因组DNA拷贝数的改变存在差异,有望通过进一步扩大样本的深入研究建立起能够鉴别两者的分子模型。
第二章:HSPC300基因异常表达对非小细胞肺癌转移潜能影响的研究
在本项研究前期工作构建的肺癌相关差异表达cDNA文库中,HSPC300是表达显著升高的基因之一。本课题在了解HSPC300在大样本非小细胞肺癌组织中表达情况的基础上分析此基因与肿瘤转移的相关性,继而进一步以肺癌细胞系模型探讨其参与肿瘤转移的可能分子机制。
针对肺癌组织微阵列(含来自128例肺鳞癌患者的162个组织样品)的免疫组织化学分析显示,与对照组织比较,HSPC300在非小细胞肺癌患者的肿瘤组织中表达显著升高(p＜0.0001),且在发生淋巴结转移的原发瘤与无淋巴结转移的原发瘤之间存在显著差异(p=0.0001),但转移瘤与原发瘤之间差异不明显(p=0.063)。此外,HSPC300的表达与临床分期也存在显著的相关性。在随后的体外实验中,用RNAi技术沉默HSPC300基因在肺癌细胞系PG中的表达,观察对高转移潜能肺癌细胞表型的影响。结果显示,沉默HSPC300基因会导致PG细胞胞膜上突起和伪足减少,迁移能力减弱。免疫共沉淀与免疫荧光分析提示,HSPC300可能与WAVE1和WAVE2存在蛋白质相互作用。HSPC300蛋白丰度的降低会导致WAVE1和WAVE2发生蛋白酶体依赖性的降解。分别沉默HSPC300和WAVE2基因均会改变PG细胞形态,同时阻断EGF经Rac至F-actin之间的信号传递;而沉默WAVE1基因则对细胞形态没有明显影响。
上述研究资料提示,与非小细胞肺癌转移相关的蛋白HSPC300主要通过影响细胞迁移相关蛋白WAVE2的稳定性来调节F-actin的形成,从而通过影响细胞骨架来实现对肿瘤转移的促进作用。
ChapterⅠ:Distinguishing non-invasive and invasive bladder urothelial carcinomas by molecular markers selected from genomic DNA
The differences between two subtypes of bladder urothelial carcinoma,non-invasive tumor and invasive tumor,are closely associated with the clinical performance,the surgical style and the prognoses.However there is a lack of reliable approaches to preoperative differentiation for the two subtypes of bladder cancer.In this study, array comparative genome hybridization(array CGH) analysis was performed first,to identify the DNA copy number variation(CNV) profiles that specifically related to the non-invasive or invasive bladder cancer,respectively.From these,a set of gain or lose genes/regions could be identified to distinguish the two subtypes of tumor. Based on subsequent validation by quantitative PCR and bioinformatical analysis,a panel of molecular markers was selected for classifying the two subtypes of bladder cancer.
The tumor tissue samples derived from 35 patients of bladder cancer(18 non-invasive and 17 invasive) were screened by array CGH on the Agilent Human Genome CGH 44B array.With analyses by CGH Analytics 3.4(Agilent) and R project (Bioconductor),the genome-wide CNV profiles of bladder urothelial carcinoma were obtained.In euchromosomes,the copy number alterations were mainly occurred in 1q,2q,3p,4q,5p,5q,7p,8p,8q,9p,9q,10q,11p,11q,13q,15q,16p,16q,17p,17q and 18q.The highest average frequency of the alterations was observed on Chromosome 9.Fourteen genes/regions with high-level amplification and 11 genes/regions with homozygous deletion,whose average frequency were no less than 20%,were identified.In the invasive tumors,the frequency of aberrancy was significantly higher than that detected in the non-invasive ones,especially in Chromosome 5,8,10,17 and 18.Bioinformatical analysis was performed with the array CGH data of the 35 cases,and 8 genes that could classify the two subtypes of bladder cancer correctly(97.1%) were screened out.These 8 genes were then validated by real time polymerase chain reaction(RT-PCR) in this group of tumor tissues.There was a significant correlation of the results between the array CGH and RT-PCR analyses(p=0.0003).
In conclusion,data obtained from a reliable array CGH analysis indicated that there were differences in the genome-wide CNV profiles between the non-invasive and invasive bladder cancers.It was promised to build up a molecular model to distinguish the bladder urothelial carcinomas with various clinical phenotypes,by further investigation with an enlarged sample size.
ChapterⅡOverexpression of HSPC300 is associated with the potential of metastasis in human non-small cell lung cancer
Our previous work suggests that HSPC300 is one of most overexpressed genes in a differentially expressed cDNA library derived from human non-small cell lung cancer (NSCLC).In present study,following the expressive status of HSPC300 protein in the tumor tissues of NSCLC was examined,the correlation between expression of the HSPC300 and the tumor metastasis was analyzed.The subsequent in vitro investigation focused on the molecular mechanism that HSPC300 facilitates metastasis of NSCLC.
Tissue microarrays derived from NSCLCs were stained immunohistochemically. HSPC300 protein was detected in 82.9%(92 of 111) of the primary tumors and 73.7% (28 of 38) of lymph node metastases,but only in 30.8%(4 of 13) of normal lung or bronchial epithelium tissue samples(p＜0.0001).HSPC300 expression was associated with the tumor staging and lymph node invasion(p=0.0035 and p=0.0001, respectively).In vitro,silencing HSPC300 by siRNA could reduce the protrusions and pseudopods formed in NSCLC cells PG that has high potential of metastasis. Moreover,downregulating HSPC300 expression inhibited migration and invasion of the PG cells by Transwell assay.With co-immunoprecipitation and immunofluorescence analyses,it was observed that there was protein-protein interaction between HSPC300 and WAVE1 or WAVE2.HSPC300 affected the proteasome-dependent stability of WAVE1 and WAVE2.However,only knock-down WAVE2,but not WAVE1,resulted in morphological change that is similar to that of silencing HSPC300 in the PG cells.EGF activates F-actin to polymerization.Knock-down HSPC300 or WAVE2 abolished the activation of the polymerization.
The current findings suggest that HSPC300 may facilitate metastasis of NSCLC mainly through stabilizing WAVE2,a protein associated with cell migration.
手术前准确区分非浸润性或浸润性膀胱尿路上皮癌对于制定合理的临床治疗方案至关重要;然而,目前尚缺乏有效易行的鉴别诊断手段。本课题在建立稳定的微阵列比较基因组杂交(array comparative genome hybridization,array CGH)技术的基础上研究膀胱癌基因组DNA的异常,以期获得膀胱癌基因组DNA拷贝数改变谱型;进而揭示非浸润性与浸润性膀胱癌的特征性改变的基因或区域,并以这些DNA分子组合来区分两种类型的膀胱癌。这项研究的远期目标为,利用一组DNA分子标志物对非浸润性和浸润性膀胱癌进行手术前的辅助鉴别诊断,以指导正确治疗。
首先,应用Agilent Human Genome CGH 44B芯片,针对35例膀胱尿路上皮癌(其中非浸润性膀胱癌18例,浸润性膀胱癌17例)的组织样品基因组DNA进行了array CGH检测。继而,采用CGH Analytics 3.4(Agilent)和R project(Bioconductor)软件分析了35张芯片的实验数据。结果显示,除性染色体之外,膀胱癌组织基因组DNA拷贝数异常变化主要发生在1q、2q、3p、4q、5p、5q、7p、8p、8q、9p、9q、10q、11p、11q、13q、15q、16p、16q、17p、17q、18q等染色体臂,其中9号染色体改变的发生频率最高。而超过20%的高水平扩增区或纯合性缺失区分别为14个和11个。浸润性膀胱癌基因组DNA在5、8、10、17、18号染色体的改变显著高于非浸润性膀胱癌。经过对35例array CGH实验数据的生物信息学分析,初步筛选出8个能区分非浸润性与浸润性膀胱癌的基因(或区域)。然后以实时定量PCR技术在上述35例起始基因组DNA样品中进行验证,从而确认了array CGH分析结果的可靠性。
上述研究资料表明,在本实验室内已经建立起稳定可靠的array CGH分析技术;非浸润性与浸润性膀胱癌基因组DNA拷贝数的改变存在差异,有望通过进一步扩大样本的深入研究建立起能够鉴别两者的分子模型。
第二章:HSPC300基因异常表达对非小细胞肺癌转移潜能影响的研究
在本项研究前期工作构建的肺癌相关差异表达cDNA文库中,HSPC300是表达显著升高的基因之一。本课题在了解HSPC300在大样本非小细胞肺癌组织中表达情况的基础上分析此基因与肿瘤转移的相关性,继而进一步以肺癌细胞系模型探讨其参与肿瘤转移的可能分子机制。
针对肺癌组织微阵列(含来自128例肺鳞癌患者的162个组织样品)的免疫组织化学分析显示,与对照组织比较,HSPC300在非小细胞肺癌患者的肿瘤组织中表达显著升高(p＜0.0001),且在发生淋巴结转移的原发瘤与无淋巴结转移的原发瘤之间存在显著差异(p=0.0001),但转移瘤与原发瘤之间差异不明显(p=0.063)。此外,HSPC300的表达与临床分期也存在显著的相关性。在随后的体外实验中,用RNAi技术沉默HSPC300基因在肺癌细胞系PG中的表达,观察对高转移潜能肺癌细胞表型的影响。结果显示,沉默HSPC300基因会导致PG细胞胞膜上突起和伪足减少,迁移能力减弱。免疫共沉淀与免疫荧光分析提示,HSPC300可能与WAVE1和WAVE2存在蛋白质相互作用。HSPC300蛋白丰度的降低会导致WAVE1和WAVE2发生蛋白酶体依赖性的降解。分别沉默HSPC300和WAVE2基因均会改变PG细胞形态,同时阻断EGF经Rac至F-actin之间的信号传递;而沉默WAVE1基因则对细胞形态没有明显影响。
上述研究资料提示,与非小细胞肺癌转移相关的蛋白HSPC300主要通过影响细胞迁移相关蛋白WAVE2的稳定性来调节F-actin的形成,从而通过影响细胞骨架来实现对肿瘤转移的促进作用。
ChapterⅠ:Distinguishing non-invasive and invasive bladder urothelial carcinomas by molecular markers selected from genomic DNA
The differences between two subtypes of bladder urothelial carcinoma,non-invasive tumor and invasive tumor,are closely associated with the clinical performance,the surgical style and the prognoses.However there is a lack of reliable approaches to preoperative differentiation for the two subtypes of bladder cancer.In this study, array comparative genome hybridization(array CGH) analysis was performed first,to identify the DNA copy number variation(CNV) profiles that specifically related to the non-invasive or invasive bladder cancer,respectively.From these,a set of gain or lose genes/regions could be identified to distinguish the two subtypes of tumor. Based on subsequent validation by quantitative PCR and bioinformatical analysis,a panel of molecular markers was selected for classifying the two subtypes of bladder cancer.
The tumor tissue samples derived from 35 patients of bladder cancer(18 non-invasive and 17 invasive) were screened by array CGH on the Agilent Human Genome CGH 44B array.With analyses by CGH Analytics 3.4(Agilent) and R project (Bioconductor),the genome-wide CNV profiles of bladder urothelial carcinoma were obtained.In euchromosomes,the copy number alterations were mainly occurred in 1q,2q,3p,4q,5p,5q,7p,8p,8q,9p,9q,10q,11p,11q,13q,15q,16p,16q,17p,17q and 18q.The highest average frequency of the alterations was observed on Chromosome 9.Fourteen genes/regions with high-level amplification and 11 genes/regions with homozygous deletion,whose average frequency were no less than 20%,were identified.In the invasive tumors,the frequency of aberrancy was significantly higher than that detected in the non-invasive ones,especially in Chromosome 5,8,10,17 and 18.Bioinformatical analysis was performed with the array CGH data of the 35 cases,and 8 genes that could classify the two subtypes of bladder cancer correctly(97.1%) were screened out.These 8 genes were then validated by real time polymerase chain reaction(RT-PCR) in this group of tumor tissues.There was a significant correlation of the results between the array CGH and RT-PCR analyses(p=0.0003).
In conclusion,data obtained from a reliable array CGH analysis indicated that there were differences in the genome-wide CNV profiles between the non-invasive and invasive bladder cancers.It was promised to build up a molecular model to distinguish the bladder urothelial carcinomas with various clinical phenotypes,by further investigation with an enlarged sample size.
ChapterⅡOverexpression of HSPC300 is associated with the potential of metastasis in human non-small cell lung cancer
Our previous work suggests that HSPC300 is one of most overexpressed genes in a differentially expressed cDNA library derived from human non-small cell lung cancer (NSCLC).In present study,following the expressive status of HSPC300 protein in the tumor tissues of NSCLC was examined,the correlation between expression of the HSPC300 and the tumor metastasis was analyzed.The subsequent in vitro investigation focused on the molecular mechanism that HSPC300 facilitates metastasis of NSCLC.
Tissue microarrays derived from NSCLCs were stained immunohistochemically. HSPC300 protein was detected in 82.9%(92 of 111) of the primary tumors and 73.7% (28 of 38) of lymph node metastases,but only in 30.8%(4 of 13) of normal lung or bronchial epithelium tissue samples(p＜0.0001).HSPC300 expression was associated with the tumor staging and lymph node invasion(p=0.0035 and p=0.0001, respectively).In vitro,silencing HSPC300 by siRNA could reduce the protrusions and pseudopods formed in NSCLC cells PG that has high potential of metastasis. Moreover,downregulating HSPC300 expression inhibited migration and invasion of the PG cells by Transwell assay.With co-immunoprecipitation and immunofluorescence analyses,it was observed that there was protein-protein interaction between HSPC300 and WAVE1 or WAVE2.HSPC300 affected the proteasome-dependent stability of WAVE1 and WAVE2.However,only knock-down WAVE2,but not WAVE1,resulted in morphological change that is similar to that of silencing HSPC300 in the PG cells.EGF activates F-actin to polymerization.Knock-down HSPC300 or WAVE2 abolished the activation of the polymerization.
The current findings suggest that HSPC300 may facilitate metastasis of NSCLC mainly through stabilizing WAVE2,a protein associated with cell migration.
引文
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