基因芯片联合组织芯片初步探讨Barrett食管发生发展的分子机制
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摘要
背景和目的:
Barrett食管(Barrett's esophagus, BE)是指食管下段复层鳞状上皮被化生的单层柱状上皮所替代的一种病理现象,可伴肠化或无肠化。其中伴有特殊肠上皮化生者属于食管腺癌(Esophagus adenocarcinoma, EAC)的癌前病变。近年来研究表明,EAC的发病率逐年上升,而食管鳞癌( Esophagus squamous carcinoma, ESC)的发病率则有下降趋势。BE已被证实与EAC的发生密切相关,超过80%的EAC发生于BE。随着现代生物技术的快速发展,BE的基础与临床研究取得了长足的进步,但其确切的分子机制尚不清楚。组织芯片技术和基因芯片以及传统组织学技术的结合,为肿瘤组织及其癌前病变中差异表达基因及其编码蛋白产物的鉴定和性质的确定提供了一种强有力的策略,利用生物芯片技术分析BE黏膜及其周围鳞状上皮的差异基因和蛋白质点,从RNA和蛋白质水平对BE发病和恶变机理进行研究,目前尚较少报道。本研究拟联合基因芯片和组织芯片鉴定BE发生发展过程中的差异表达基因,考察相关差异表达基因与BE临床病理特征之间以及基因相互之间的相关性,揭示BE发生发展的分子机制全貌,为临床诊治提供新的潜在靶点。
材料和方法
1.临床标本
BE患者来自于第三军医大学附属西南医院消化内镜中心的门诊BE患者, EAC患者来自于西南医院消化内镜中心的门诊EAC患者以及第三军医大学附属西南医院胸外科、附属新桥医院胸外科手术患者。收集患者BE黏膜以及正常食管黏膜组织、食管腺癌及癌旁组织,之前获得家属的知情同意以及医院临床研究委员会批准。组织活检后立刻储存于液氮,实验时移入-80℃冰箱。标本大小约4mm ,正常食管黏膜组织从BE黏膜上方2 cm处获取。所有病理组织样本特征均经常规H E染色证实。
2. RNA准备
组织标本在-196℃液氮中研磨成碎末,之后经Trizol(Invitrogen Life Technologies,美国)提取组织总RNA。RNA的完整性和纯度经1.2%甲醇琼脂糖凝胶电泳验证。总RNA纯度OD260/OD280 > 1.8用于后续基因芯片分析。
3.标记和杂交
将来自于BE及其相应的食管粘膜组织的总RNA 20μg通过Micromax Direct labeling试剂盒(Perkin Elmer Life Sciences, USA)标记上Cy3和Cy5。将标记好的探针于95℃变性5分钟,然后在杂交小室内((Corning Life Sciences, USA))65℃水浴18小时,将其杂交到包含30,968探针的Agilent寡核苷酸芯片上。在杂交之前先将芯片先在5XSSC, 0.1% SDS和1% BSA溶液中65℃水浴45分钟预杂交,以减少非特异性杂交。杂交之后,将芯片相继在含0.1% SDS的2XSSC、含0.05% SDS的1XSSC及0.1XSSC溶液中洗涤20分钟,之后将芯片烤干。
4.芯片图像分析
芯片经Gene Pix 4200A扫描仪(Axon Instruments Inc. Foster City, CA)在不同的PMT设置下进行扫描,以获得最大的信号浓度并且探针溶解率<0.1%。Cy5标记的cRNA在635 nm波长下扫描,Cy3标记的cRNA样品在532 nm扫描。扫描结果以TIFF图像格式保存并经Gene Pix Pro 6.0.1.27软件(Axon Instrument)分析。所得到的图像为两种荧光的复合图,须经Split-tiff软件分割成单色荧光图像。将图像导入图像分析软件Imagene,经过自动和人工定位与排列,确定杂交点的范围,过滤背景噪音,提取得到基因表达的荧光信号强度值,最后以列表形式输出,从而完成将扫描得到的图像定量转化为数值。杂交图是经过计算机数据叠加后产生的图像,反映了每个被检测基因在两者相互比较中表达丰度的比值。采用Agilent扫描仪进行扫描,差异基因筛选标准为ratio≥2为上调基因,ratio≤0.5为下调基因。红色点代表该基因在BE组织中表达上调,绿色点代表该基因在BE中表达下调,而黄色点代表对比组织间没有差异。
5.生物信息分析
聚类分析采用Michael Eisen编著的Cluster Software version 3.0软件进行分析。所有基因表达数据经log转换。将结果经Tree View Program Version 1.50软件进行分析和可视化。上调基因和下调基因分别用红色和绿色代表。KEEG Pathway分析:应用免费基因分类和数据库得到生物过程、分子功能及细胞定位等功能节点。根据免费的KEGG数据库以及Biointerpreter software (http://www.genotypic.co.in/biointerpreter)得到基因在已知生物通路中的作用。
6.实时荧光定量RT-PCR
用LC定量PCR仪(Roche Diagnostics)及TaqMan SYBR GreenⅠ(RocheLaboratories)荧光法分析了5个上调基因(FLJ45831、MUC1、LYN、ITGβ1、RNF121)和2个下调基因(CFL1、RPS6)的表达情况;以GAPDH作为管家基因。目的基因经RT-PCR试剂盒(Invitrogen)检测。经过45个循环扩增后,得到每个孔相对于原始浓度的Ct值,这些原始数据经过ABI Prism7000 SDS软件进行分析,计算均值和标准差。
7.组织芯片(TMA)
TMA的构建根据TMA构建流程进行。样品经过脱水和HE染色后,不同的组织样品固定在芯片上。通过免疫组织化学(IHC)检测目的蛋白在BE, EAC and ESC上的表达。三种目的蛋白Cofilin, Inergrin beta 1和RNF121通过TMA技术检测。
8. Western-Blot
组织蛋白经BCA蛋白提取试剂盒提取。50μg蛋白和2×上样buffer变性后经SDS-PAGE电泳分离,然后转移到硝酸纤维素膜上。通过特异性抗体及相应二抗进行染色,用化学发光法进行显色。
9.统计学分析
所有数据以均值±SD表示,配对资料经双尾t检验,P<0.05有统计学意义。
结果
1.总RNA的抽提和mRNA的纯化
总RNA经琼脂糖凝胶电泳得到A260/A280在1.843 - 1.951之间,mRNA在1.90 - 2.0,每条泳道可见28S和18S条带,证明RNA的浓度和纯度均较高,符合实验要求。
2.基因芯片检测到差异表达基因BE基因芯片结果显示,共计426个基因在BE形成过程中发生明显改变,其中上调基因共142个,下调基因共284个。这些基因涉及粘蛋白相关基因、癌基因与抑癌基因、骨形态蛋白、凋亡抑制基因和bcl-2家族相关基因等。基因分类和KEGG途径分析发现四种分子功能途径在BE中表达上调,这四种途径包括钾离子通道活化、活性钙钾离子通道活化,RNA多聚酶II启动子转录的负调控,过渡金属运输途径。许多基因家族在BE中的表达较正常粘膜增高,这些家族包括FLJ45831蛋白、钾离子介导活性钙通道、癌胚抗原相关性细胞粘附分子1(胆汁糖化蛋白)、淋巴细胞毒素β(肿瘤坏死因子超家族3)、载脂蛋白B48受体、胃内因子(维生素B合成物)、MBT结构域1、BRCA1基因2等。
EAC基因芯片结果显示,在2倍差异表达基因中,上调基因共212个,下调基因共126个,涉及了细胞周期相关基因、信号转导相关基因、肿瘤转移相关基因、血管生成相关基因、细胞增殖相关基因、凋亡抑制基因、抑癌基因、黏附和代谢等。
3.实时荧光定量RT-PCR对基因芯片的验证我们对经基因芯片刷选的7个基因进行了验证,结果发现在两种检测方法不仅在表达趋势上相似,而且在表达水平上也很相近。
4. TMA检测Cofilin、Intergrinβ1、RNF121的表达TMA检测发现Cofilin、Intergrinβ1、RNF121在BE中的表达均较食管粘膜表达增高,在EAC和ESC上的表达较癌旁组织升高,并且在BE中的表达较EAC降低。
5. Western-Blot检测Cofilin和RNF121的表达结构发现Cofilin和RNF121在BE中的表达均较食管粘膜表达增高,在EAC中的表达较癌旁组织升高,并且在BE中的表达较EAC降低。这和TMA检测结果相一致。
结论
1.大活检钳钳取2次活检的组织能够提供芯片上样量所需要的5ugRNA,基因芯片分析可用于内镜下活检组织;
2. BE在基因和分子生物水平上的改变比组织学改变发生要早,是Barrett腺癌的癌前病变。Barrett食管的发生发展涉及多基因多步骤,是一个复杂的过程,这些差异表达基因可能与BE的发生、发展以及向EAC的转化有关。
3. MMP相关基因表达上调、CYP相关基因表达下调及CYP2亚家族基因多态性可能涉及食管腺癌的发生、发展过程。
4.寡核苷酸芯片和实时荧光定量分析基因表达变化都是可信的,基因芯片筛选基因表达谱具有高通量大规模的特点,而实时荧光定量RT-PCR则适合单个基因表达变化的研究,两者互为补充和验证。
5. TMA技术发现Cofilin、Intergrinβ1及RNF121在BE中表达较正常食管粘膜均增高,并且在EAC及ESC中的表达较癌旁组织升高,表明Cofilin、Intergrinβ1和RNF121可能在BE发生及发展为EAC的过程中起着一定的作用。
6. Western-Blot检测结果与TMA相一致,可见组织芯片技术与Western技术相结合,可以组成一个可靠的高通量蛋白表达分析系统;与基因芯片相结合就可以形成完整的基因表达、扩增和功能检测分析系统。这为深入理解BE发生、发展的分子机制提供了有力的实验依据。
Backgrouds
Barrett’s esophagus (Barrett’s metaplasia, BE) is characterized by specialized metaplastic intestinal epithelium replacing the normal squamous epithelium in the distal esophagus. Barrett's esophagus is thought to be a premalignant transformation and has been identified in 80% to 100% of esophageal adenocarcinoma of the distal esophagus(EAC). A metaplasia-dysplasia-carcinoma sequence links Barrett’s esophagus with EAC, which is one of the fastest-increasing cancers in the Western world. Patients with Barrett's oesophagus have a 2-25% risk of developing mild to severe dysplasia and a 2-5% risk of having adenocarcinoma: 30-150 times higher than the risk in the general population. Forty to fifty percent of Barrett's esophagus patients with severe dysplasia would present adenocarcinoma within 5 years[2]. Along with the progression of biotechnology, the study of the mechanisms of BE got great improvement. However, its exact molecular mechanism was still unclear.
Recent advances in biotechnology have revolutionized the high-throughput analysis of gene expression. In particular, the development of gene chip technologies has made it possible to analyse the expression of thousands of genes simultaneously. gene chip technology, in combination with bioinformatics analyses, promises more accurate disease classification, earlier detection, and higher efficiency in the field of cancer diagnosis[4].Gene expression profiling studies have been used in the examination of cancer progression, diagnosis, drug target discovery, and gene therapy evaluation. Many studies have applied this technology for Barrett's metaplasia and adenocarcinoma, and identified a number of candidate genes useful as biomarkers in cancer staging, prediction of recurrence,prognosis, and personalized therapy. Some of these target molecules have been used to develop new serum diagnostic markers and therapeutic targets against BE to benefit patients. Functional classification of differentially expressed genes was then performed to discover molecular pathways and subgroupings associated with each carcinogenic transition. Furthermore, we examined global gene signatures as a potential means of risk stratification at precancerous stages[5].On the other hand, the tissue micro array (TMA)technology can simultaneously detect decades even thousands samples on one chip in situ. As an important branch of biochip, TMA has become a key tool for promoting the quickly transformation of the study results of molecular genetics, genomics, proteome to clinical application. Following by the enforcement of postgenome project, TMA provided an unprecedented high performance methods for discovery biological behaviour, molecular diagnosis, prognosis assessment, and individualized treatment related to tumors. It will be an great contribution to realizing the nature of tumor and finding effective therapy.
To identify the molecules involved in Barrett's metaplasia and carcinogenesis and for the development of new molecular therapies, we performed gene expression profile analysis using a cDNA microarray, and detected some related proteins by TMA.
Materials and Methods
1. Clinical samples
Endoscopic tissue biopsy specimens were taken from BE patients at Gastroenterology Research Institute,Southwest Hospital,Third Military Medical University,Chongqing, China. Routine histopathologic analysis was done to confirm the diagnosis by experienced gastrointestinal pathologists. These samples were labeled and snap frozen in liquid nitrogen and stored at–80°C for future RNA extraction. Informed consent was obtained from all patients. Data of clinicopathologic parameters were obtained from patients’clinical records and pathologic reports. Institutional Human Ethics Committee approved the study.
2. RNA preparation
Tissues were ground into powder in -196℃liquid nitrogen and homogenized using Trizol reagent (Invitrogen Life Technologies, CA) for extraction of total RNA following the instruction of the manufacturer. The integrity of total RNA was checked by 1.2% formaldehyde agarose gel electrophoresis (visual presence of 28S and 18S bands). Total RNA with OD260/OD280 > 1.8 was used for microarray experiments.
3. Labeling and hybridization
Twentyμg of total RNA from the tumor and matched normal tissue were labeled with cyanine 3-dUTP and cyanine 5-dUTP by direct labeling method (Perkin Elmer Life Sciences, USA: Micromax Direct labeling kit). The labeled probes were denatured at 95℃for 5 min and hybridized with a human Agilent oligomicroarray(30,968probes) in a hybridization chamber (Corning Life Sciences, USA) at 65℃water bath for 18 h. Before hybridization, slides were pre-hybridized in 5XSSC, 0.1% SDS and 1% BSA solution at 65℃for 45 min to prevent nonspecific hybridization. After hybridization, the slides were washed in 2XSSC with 0.1% SDS, 0.1X SSC with 0.05% SDS and 0.1XSSC sequentially for 20 min each and then spin-dried.
4. Microarray image analysis
Hybridized arrays were scanned at 5 mm resolution on a Gene Pix 4200A scanner (Axon Instruments Inc. Foster City, CA) at various PMT voltage settings to obtain maximal signal intensities with < 0.1% probe saturation. The Cy5-labelled cRNAs were scanned at 635 nm and the Cy3-labeled cRNA samples were scanned at 532 nm. The resulting TIFF images were analyzed by Gene Pix Pro 6.0.1.27 software (Axon Instrument). Both digital images were overlaid to form a pseudo colored image and a detection method was then used to determine the actual target region based on the information from both red (Cy5) and green (Cy3) pixel values. The ratios of the sample intensity to the reference intensity (green: red) for all of the targets were determined and ratio normalization was performed to normalize the center of the ratio distribution to 1.0. Image processing analysis was used for estimation of spot quality by assigning a quality score to each ratio measurement[8]. Data were acquired using MAS 5.0 software (Agilent) and exported to MS Excel.
5. Bioinformatics analysis
Hierarchical clustering: Average linkage hierarchical clustering was done using the Cluster Software version 3.0 written by Michael Eisen[11]. The Euclidean distance metric was used as a measure of similarity between the gene expression patterns for each pair of samples based on log-transformed ratios across all genes. The results were analyzed and visualized with the Tree View Program Version 1.50 also written by Michael Eisen. Those genes showing progressive fold increases or decreases in gene expression relative to normal mucosa were shown proportionally in red and green, respectively.
Pathway prediction analysis: We obtained annotations of the bioprocesses, molecular function and cellular localization using the freely available Gene Ontology and Source database[12].The significant gene clusters were queried with known components of the biological pathways on the freely available KEGG database[13]. We also used the Biointerpreter software (http://www.genotypic.co.in/biointerpreter) for gene ontology.
6. Real-time RT-PCR
The expressions of 5 up-regulated genes(FLJ45831、MUC1、LYN、ITGβ1、RNF121) and 2 down-regulated genes (CFL1、RPS6)were analyzed by TaqMan SYBR GreenⅠ(Roche Laboratories) fluorescence and LC quantitative PCR(Roche Diagnostics),using GAPDH as a house-keeping gene. The target genes were amplified according to the instruction of Invitrogen RT-PCR kits. After 45 cycles, the cycle threshold(Ct) of each tube and the relative original concentration C were first obtained,then these original data was analyzed by ABI Prism7000 SDS software. Through the relative original concentration ratio, the average value and standard deviation(SD) were calculated.
7. TMA
The construction of TMA was performed according by the manuscript of TMA. The samples were dehydrolysised and died by HE, then different tissue samples were arrayed and fixed on the chip. The expression of different proteins on BE, EAC and ESC were detected by immunohistochemistry (IHC) using specific antibody. Three interested proteins Cofilin, Inergrin beta 1 and RNF121 were detected by TMA.
8. Western-Blot
The tissue protein was extracted by BCA protein assay kit according to the manuscript. 50μg proteins were denatured in 2×loading buffer at 100°C for 5min, separated on SDS-PAGE gel, and transferred onto nitrocellulose membrane. The proteins were then detected using specific antibodies and appropriate secondary antibodies and visualized by radioautography using ECL. The interested proteins Cofilin and RNF121 were detected by Western-Blot.
9. Statistical analysis
Data were expressed as mean±SD of three or more independent measurements. Paired data were subjected to two-tailed Students t test. A P value less than 0. 05 was considered statistically significant.
Results
1. Extraction of Total RNA and Purification of mRNA RNA quality of BE and normal esophageal epithelium was assessed by A260/ A280. These results showed that A260/A280 of total RNA was 1.843 - 1.951 , and that of mRNA was 1.90 - 2.0. RNA purity was examined by electrophoresis. Clear signal appearance of 28S and 18S of rRNA was seen on the electrophorogram of total RNA , suggesting no degradation in RNA.
2. Differentially Expressed Genes
The image of gene expression profiles in BE was shown in fig. 2,3. From the original number of 30,968 gene probes, 426 genes had a quality score of P <0 .05 and were subjected to further comparison. Of the 426 genes that had significantly different expression between BE and normal esophagus, 142 were upregulated and 284 were downregulated in BE. On the basis of Gene Ontology and KEGG Pathway, four different molecular functional pathways (cobalamin transport, cobalt ion transport, negative regulation of transcription from RNA polymerase II promoter, transition metal ion transport) were most significantly upregulated and six different molecular functional pathways (MAPK signaling pathway, T cell receptor signaling pathway, Ribosome, Wnt signaling pathway, VEGF signaling pathway, Apoptosis) were most significantly downregulated. The reverse transcription-PCR confirmed the results of microarrays. These genes were listed on table 1. These differentially expressed genes were classified based on Gene Ontology (GO) system and TreeView.
Many EAC-assoeiated genes were screened by the high-throughput gene chip method.There were 212 up-regulated genes and 126 down-regulated genes am- ong 2-fold DEGs. including 16 genes related to cytochrome P450 (CYP)
3. Confirmation by Real time RT PCR
The arrays screening results of 7 genes were conformed by real-time RT-PCR . We found that there was a good correlation between this two methods. The results of 7 target genes were not only consistent with the results of Agilent gene hybridization, but the gene expression levels were also very similar.
4. The expression of Cofilin, Intergrinβ1,RNF121 detected by TMA
The expression of Cofilin,Intergrinβ1,RNF121in BE were all significantly increased compared to normal esophagus mucous, and their expression in EAC and ESC were increased than in latero-cancer tissues. Furthermore, The expression of Cofilin,Intergrinβ1,RNF121in EAC were all significantly increased compared to in BE,but not in EAC.
5. The expression of Cofilin and RNF121 detected by Western-Blot
The expression of Cofilin and RNF121 in BE were both significantly increased compared to normal esophagus mucous, and their expression in EAC and ESC were increased than in latero-cancer tissues. Furthermore, The expression of Cofilin, RNF121in EAC were all significantly increased compared to in BE,but not in EAC. These results were matched to the results of TMA.
Conclusion
1. Two biopsies by disposable jumbo biopsy forceps provided approximately 5 microg required for microarrays. Microarray-based studies are feasible in endoscopically obtained tissues.
2. 426 different expressed genes including 142 up-regulated genes and 284 down-regulated genes were screened by gene chip. These genes involved in cell cycle, signal transduction, mucoprotein, oncogene and anti-oncogene, bone morphous protein, apoptosis inhibiting, and BCL-2 family. These different expressed genes maybe related to the genesis and development of BE, and at the same time these genes maybe related to the transformation of BE to EAC.
3. The up-regulated expression of MMP related genes、down-regulated expression of CYP related genes and gene polymorphism of CYP2 subfamily may be involved in the onset and progress of EAC.
4. The results of Real-time RT-PCR were matched to that of gene chip and both gene chip and Real-time RT-PCR were credible. Gene chip has the feature of high flux and large scale, but the Real-time RT-PCR was suit for the study of single gene. They are mutual for supplement and verification.
5. It was discovered by TMA that the expression of Cofilin ,Intergrinβ1and RNF121 were all increased in BE,EAC and ESC, and The expression of Cofilin,Intergrinβ1,RNF121in EAC were all significantly increased compared to in BE,but not in EAC. It is demonstrated that Cofilin、Intergrinβ1and RNF121 were related to the genesis of BE and played a great role for BE transformed to EAC. This was an original discovery for the mechanism of BE.
6. The results of Western-blot were matched to that of TMA. It demonstrated that TMA was a reliable and high flux protein analytical system. Gene chip combined with TMA would provide an integrity analytical system for gene expression, amplification and function detection. The results we got provided vigorous evidence for deeply understanding the genesis and improvement of BE.
Barrett食管(Barrett's esophagus, BE)是指食管下段复层鳞状上皮被化生的单层柱状上皮所替代的一种病理现象,可伴肠化或无肠化。其中伴有特殊肠上皮化生者属于食管腺癌(Esophagus adenocarcinoma, EAC)的癌前病变。近年来研究表明,EAC的发病率逐年上升,而食管鳞癌( Esophagus squamous carcinoma, ESC)的发病率则有下降趋势。BE已被证实与EAC的发生密切相关,超过80%的EAC发生于BE。随着现代生物技术的快速发展,BE的基础与临床研究取得了长足的进步,但其确切的分子机制尚不清楚。组织芯片技术和基因芯片以及传统组织学技术的结合,为肿瘤组织及其癌前病变中差异表达基因及其编码蛋白产物的鉴定和性质的确定提供了一种强有力的策略,利用生物芯片技术分析BE黏膜及其周围鳞状上皮的差异基因和蛋白质点,从RNA和蛋白质水平对BE发病和恶变机理进行研究,目前尚较少报道。本研究拟联合基因芯片和组织芯片鉴定BE发生发展过程中的差异表达基因,考察相关差异表达基因与BE临床病理特征之间以及基因相互之间的相关性,揭示BE发生发展的分子机制全貌,为临床诊治提供新的潜在靶点。
材料和方法
1.临床标本
BE患者来自于第三军医大学附属西南医院消化内镜中心的门诊BE患者, EAC患者来自于西南医院消化内镜中心的门诊EAC患者以及第三军医大学附属西南医院胸外科、附属新桥医院胸外科手术患者。收集患者BE黏膜以及正常食管黏膜组织、食管腺癌及癌旁组织,之前获得家属的知情同意以及医院临床研究委员会批准。组织活检后立刻储存于液氮,实验时移入-80℃冰箱。标本大小约4mm ,正常食管黏膜组织从BE黏膜上方2 cm处获取。所有病理组织样本特征均经常规H E染色证实。
2. RNA准备
组织标本在-196℃液氮中研磨成碎末,之后经Trizol(Invitrogen Life Technologies,美国)提取组织总RNA。RNA的完整性和纯度经1.2%甲醇琼脂糖凝胶电泳验证。总RNA纯度OD260/OD280 > 1.8用于后续基因芯片分析。
3.标记和杂交
将来自于BE及其相应的食管粘膜组织的总RNA 20μg通过Micromax Direct labeling试剂盒(Perkin Elmer Life Sciences, USA)标记上Cy3和Cy5。将标记好的探针于95℃变性5分钟,然后在杂交小室内((Corning Life Sciences, USA))65℃水浴18小时,将其杂交到包含30,968探针的Agilent寡核苷酸芯片上。在杂交之前先将芯片先在5XSSC, 0.1% SDS和1% BSA溶液中65℃水浴45分钟预杂交,以减少非特异性杂交。杂交之后,将芯片相继在含0.1% SDS的2XSSC、含0.05% SDS的1XSSC及0.1XSSC溶液中洗涤20分钟,之后将芯片烤干。
4.芯片图像分析
芯片经Gene Pix 4200A扫描仪(Axon Instruments Inc. Foster City, CA)在不同的PMT设置下进行扫描,以获得最大的信号浓度并且探针溶解率<0.1%。Cy5标记的cRNA在635 nm波长下扫描,Cy3标记的cRNA样品在532 nm扫描。扫描结果以TIFF图像格式保存并经Gene Pix Pro 6.0.1.27软件(Axon Instrument)分析。所得到的图像为两种荧光的复合图,须经Split-tiff软件分割成单色荧光图像。将图像导入图像分析软件Imagene,经过自动和人工定位与排列,确定杂交点的范围,过滤背景噪音,提取得到基因表达的荧光信号强度值,最后以列表形式输出,从而完成将扫描得到的图像定量转化为数值。杂交图是经过计算机数据叠加后产生的图像,反映了每个被检测基因在两者相互比较中表达丰度的比值。采用Agilent扫描仪进行扫描,差异基因筛选标准为ratio≥2为上调基因,ratio≤0.5为下调基因。红色点代表该基因在BE组织中表达上调,绿色点代表该基因在BE中表达下调,而黄色点代表对比组织间没有差异。
5.生物信息分析
聚类分析采用Michael Eisen编著的Cluster Software version 3.0软件进行分析。所有基因表达数据经log转换。将结果经Tree View Program Version 1.50软件进行分析和可视化。上调基因和下调基因分别用红色和绿色代表。KEEG Pathway分析:应用免费基因分类和数据库得到生物过程、分子功能及细胞定位等功能节点。根据免费的KEGG数据库以及Biointerpreter software (http://www.genotypic.co.in/biointerpreter)得到基因在已知生物通路中的作用。
6.实时荧光定量RT-PCR
用LC定量PCR仪(Roche Diagnostics)及TaqMan SYBR GreenⅠ(RocheLaboratories)荧光法分析了5个上调基因(FLJ45831、MUC1、LYN、ITGβ1、RNF121)和2个下调基因(CFL1、RPS6)的表达情况;以GAPDH作为管家基因。目的基因经RT-PCR试剂盒(Invitrogen)检测。经过45个循环扩增后,得到每个孔相对于原始浓度的Ct值,这些原始数据经过ABI Prism7000 SDS软件进行分析,计算均值和标准差。
7.组织芯片(TMA)
TMA的构建根据TMA构建流程进行。样品经过脱水和HE染色后,不同的组织样品固定在芯片上。通过免疫组织化学(IHC)检测目的蛋白在BE, EAC and ESC上的表达。三种目的蛋白Cofilin, Inergrin beta 1和RNF121通过TMA技术检测。
8. Western-Blot
组织蛋白经BCA蛋白提取试剂盒提取。50μg蛋白和2×上样buffer变性后经SDS-PAGE电泳分离,然后转移到硝酸纤维素膜上。通过特异性抗体及相应二抗进行染色,用化学发光法进行显色。
9.统计学分析
所有数据以均值±SD表示,配对资料经双尾t检验,P<0.05有统计学意义。
结果
1.总RNA的抽提和mRNA的纯化
总RNA经琼脂糖凝胶电泳得到A260/A280在1.843 - 1.951之间,mRNA在1.90 - 2.0,每条泳道可见28S和18S条带,证明RNA的浓度和纯度均较高,符合实验要求。
2.基因芯片检测到差异表达基因BE基因芯片结果显示,共计426个基因在BE形成过程中发生明显改变,其中上调基因共142个,下调基因共284个。这些基因涉及粘蛋白相关基因、癌基因与抑癌基因、骨形态蛋白、凋亡抑制基因和bcl-2家族相关基因等。基因分类和KEGG途径分析发现四种分子功能途径在BE中表达上调,这四种途径包括钾离子通道活化、活性钙钾离子通道活化,RNA多聚酶II启动子转录的负调控,过渡金属运输途径。许多基因家族在BE中的表达较正常粘膜增高,这些家族包括FLJ45831蛋白、钾离子介导活性钙通道、癌胚抗原相关性细胞粘附分子1(胆汁糖化蛋白)、淋巴细胞毒素β(肿瘤坏死因子超家族3)、载脂蛋白B48受体、胃内因子(维生素B合成物)、MBT结构域1、BRCA1基因2等。
EAC基因芯片结果显示,在2倍差异表达基因中,上调基因共212个,下调基因共126个,涉及了细胞周期相关基因、信号转导相关基因、肿瘤转移相关基因、血管生成相关基因、细胞增殖相关基因、凋亡抑制基因、抑癌基因、黏附和代谢等。
3.实时荧光定量RT-PCR对基因芯片的验证我们对经基因芯片刷选的7个基因进行了验证,结果发现在两种检测方法不仅在表达趋势上相似,而且在表达水平上也很相近。
4. TMA检测Cofilin、Intergrinβ1、RNF121的表达TMA检测发现Cofilin、Intergrinβ1、RNF121在BE中的表达均较食管粘膜表达增高,在EAC和ESC上的表达较癌旁组织升高,并且在BE中的表达较EAC降低。
5. Western-Blot检测Cofilin和RNF121的表达结构发现Cofilin和RNF121在BE中的表达均较食管粘膜表达增高,在EAC中的表达较癌旁组织升高,并且在BE中的表达较EAC降低。这和TMA检测结果相一致。
结论
1.大活检钳钳取2次活检的组织能够提供芯片上样量所需要的5ugRNA,基因芯片分析可用于内镜下活检组织;
2. BE在基因和分子生物水平上的改变比组织学改变发生要早,是Barrett腺癌的癌前病变。Barrett食管的发生发展涉及多基因多步骤,是一个复杂的过程,这些差异表达基因可能与BE的发生、发展以及向EAC的转化有关。
3. MMP相关基因表达上调、CYP相关基因表达下调及CYP2亚家族基因多态性可能涉及食管腺癌的发生、发展过程。
4.寡核苷酸芯片和实时荧光定量分析基因表达变化都是可信的,基因芯片筛选基因表达谱具有高通量大规模的特点,而实时荧光定量RT-PCR则适合单个基因表达变化的研究,两者互为补充和验证。
5. TMA技术发现Cofilin、Intergrinβ1及RNF121在BE中表达较正常食管粘膜均增高,并且在EAC及ESC中的表达较癌旁组织升高,表明Cofilin、Intergrinβ1和RNF121可能在BE发生及发展为EAC的过程中起着一定的作用。
6. Western-Blot检测结果与TMA相一致,可见组织芯片技术与Western技术相结合,可以组成一个可靠的高通量蛋白表达分析系统;与基因芯片相结合就可以形成完整的基因表达、扩增和功能检测分析系统。这为深入理解BE发生、发展的分子机制提供了有力的实验依据。
Backgrouds
Barrett’s esophagus (Barrett’s metaplasia, BE) is characterized by specialized metaplastic intestinal epithelium replacing the normal squamous epithelium in the distal esophagus. Barrett's esophagus is thought to be a premalignant transformation and has been identified in 80% to 100% of esophageal adenocarcinoma of the distal esophagus(EAC). A metaplasia-dysplasia-carcinoma sequence links Barrett’s esophagus with EAC, which is one of the fastest-increasing cancers in the Western world. Patients with Barrett's oesophagus have a 2-25% risk of developing mild to severe dysplasia and a 2-5% risk of having adenocarcinoma: 30-150 times higher than the risk in the general population. Forty to fifty percent of Barrett's esophagus patients with severe dysplasia would present adenocarcinoma within 5 years[2]. Along with the progression of biotechnology, the study of the mechanisms of BE got great improvement. However, its exact molecular mechanism was still unclear.
Recent advances in biotechnology have revolutionized the high-throughput analysis of gene expression. In particular, the development of gene chip technologies has made it possible to analyse the expression of thousands of genes simultaneously. gene chip technology, in combination with bioinformatics analyses, promises more accurate disease classification, earlier detection, and higher efficiency in the field of cancer diagnosis[4].Gene expression profiling studies have been used in the examination of cancer progression, diagnosis, drug target discovery, and gene therapy evaluation. Many studies have applied this technology for Barrett's metaplasia and adenocarcinoma, and identified a number of candidate genes useful as biomarkers in cancer staging, prediction of recurrence,prognosis, and personalized therapy. Some of these target molecules have been used to develop new serum diagnostic markers and therapeutic targets against BE to benefit patients. Functional classification of differentially expressed genes was then performed to discover molecular pathways and subgroupings associated with each carcinogenic transition. Furthermore, we examined global gene signatures as a potential means of risk stratification at precancerous stages[5].On the other hand, the tissue micro array (TMA)technology can simultaneously detect decades even thousands samples on one chip in situ. As an important branch of biochip, TMA has become a key tool for promoting the quickly transformation of the study results of molecular genetics, genomics, proteome to clinical application. Following by the enforcement of postgenome project, TMA provided an unprecedented high performance methods for discovery biological behaviour, molecular diagnosis, prognosis assessment, and individualized treatment related to tumors. It will be an great contribution to realizing the nature of tumor and finding effective therapy.
To identify the molecules involved in Barrett's metaplasia and carcinogenesis and for the development of new molecular therapies, we performed gene expression profile analysis using a cDNA microarray, and detected some related proteins by TMA.
Materials and Methods
1. Clinical samples
Endoscopic tissue biopsy specimens were taken from BE patients at Gastroenterology Research Institute,Southwest Hospital,Third Military Medical University,Chongqing, China. Routine histopathologic analysis was done to confirm the diagnosis by experienced gastrointestinal pathologists. These samples were labeled and snap frozen in liquid nitrogen and stored at–80°C for future RNA extraction. Informed consent was obtained from all patients. Data of clinicopathologic parameters were obtained from patients’clinical records and pathologic reports. Institutional Human Ethics Committee approved the study.
2. RNA preparation
Tissues were ground into powder in -196℃liquid nitrogen and homogenized using Trizol reagent (Invitrogen Life Technologies, CA) for extraction of total RNA following the instruction of the manufacturer. The integrity of total RNA was checked by 1.2% formaldehyde agarose gel electrophoresis (visual presence of 28S and 18S bands). Total RNA with OD260/OD280 > 1.8 was used for microarray experiments.
3. Labeling and hybridization
Twentyμg of total RNA from the tumor and matched normal tissue were labeled with cyanine 3-dUTP and cyanine 5-dUTP by direct labeling method (Perkin Elmer Life Sciences, USA: Micromax Direct labeling kit). The labeled probes were denatured at 95℃for 5 min and hybridized with a human Agilent oligomicroarray(30,968probes) in a hybridization chamber (Corning Life Sciences, USA) at 65℃water bath for 18 h. Before hybridization, slides were pre-hybridized in 5XSSC, 0.1% SDS and 1% BSA solution at 65℃for 45 min to prevent nonspecific hybridization. After hybridization, the slides were washed in 2XSSC with 0.1% SDS, 0.1X SSC with 0.05% SDS and 0.1XSSC sequentially for 20 min each and then spin-dried.
4. Microarray image analysis
Hybridized arrays were scanned at 5 mm resolution on a Gene Pix 4200A scanner (Axon Instruments Inc. Foster City, CA) at various PMT voltage settings to obtain maximal signal intensities with < 0.1% probe saturation. The Cy5-labelled cRNAs were scanned at 635 nm and the Cy3-labeled cRNA samples were scanned at 532 nm. The resulting TIFF images were analyzed by Gene Pix Pro 6.0.1.27 software (Axon Instrument). Both digital images were overlaid to form a pseudo colored image and a detection method was then used to determine the actual target region based on the information from both red (Cy5) and green (Cy3) pixel values. The ratios of the sample intensity to the reference intensity (green: red) for all of the targets were determined and ratio normalization was performed to normalize the center of the ratio distribution to 1.0. Image processing analysis was used for estimation of spot quality by assigning a quality score to each ratio measurement[8]. Data were acquired using MAS 5.0 software (Agilent) and exported to MS Excel.
5. Bioinformatics analysis
Hierarchical clustering: Average linkage hierarchical clustering was done using the Cluster Software version 3.0 written by Michael Eisen[11]. The Euclidean distance metric was used as a measure of similarity between the gene expression patterns for each pair of samples based on log-transformed ratios across all genes. The results were analyzed and visualized with the Tree View Program Version 1.50 also written by Michael Eisen. Those genes showing progressive fold increases or decreases in gene expression relative to normal mucosa were shown proportionally in red and green, respectively.
Pathway prediction analysis: We obtained annotations of the bioprocesses, molecular function and cellular localization using the freely available Gene Ontology and Source database[12].The significant gene clusters were queried with known components of the biological pathways on the freely available KEGG database[13]. We also used the Biointerpreter software (http://www.genotypic.co.in/biointerpreter) for gene ontology.
6. Real-time RT-PCR
The expressions of 5 up-regulated genes(FLJ45831、MUC1、LYN、ITGβ1、RNF121) and 2 down-regulated genes (CFL1、RPS6)were analyzed by TaqMan SYBR GreenⅠ(Roche Laboratories) fluorescence and LC quantitative PCR(Roche Diagnostics),using GAPDH as a house-keeping gene. The target genes were amplified according to the instruction of Invitrogen RT-PCR kits. After 45 cycles, the cycle threshold(Ct) of each tube and the relative original concentration C were first obtained,then these original data was analyzed by ABI Prism7000 SDS software. Through the relative original concentration ratio, the average value and standard deviation(SD) were calculated.
7. TMA
The construction of TMA was performed according by the manuscript of TMA. The samples were dehydrolysised and died by HE, then different tissue samples were arrayed and fixed on the chip. The expression of different proteins on BE, EAC and ESC were detected by immunohistochemistry (IHC) using specific antibody. Three interested proteins Cofilin, Inergrin beta 1 and RNF121 were detected by TMA.
8. Western-Blot
The tissue protein was extracted by BCA protein assay kit according to the manuscript. 50μg proteins were denatured in 2×loading buffer at 100°C for 5min, separated on SDS-PAGE gel, and transferred onto nitrocellulose membrane. The proteins were then detected using specific antibodies and appropriate secondary antibodies and visualized by radioautography using ECL. The interested proteins Cofilin and RNF121 were detected by Western-Blot.
9. Statistical analysis
Data were expressed as mean±SD of three or more independent measurements. Paired data were subjected to two-tailed Students t test. A P value less than 0. 05 was considered statistically significant.
Results
1. Extraction of Total RNA and Purification of mRNA RNA quality of BE and normal esophageal epithelium was assessed by A260/ A280. These results showed that A260/A280 of total RNA was 1.843 - 1.951 , and that of mRNA was 1.90 - 2.0. RNA purity was examined by electrophoresis. Clear signal appearance of 28S and 18S of rRNA was seen on the electrophorogram of total RNA , suggesting no degradation in RNA.
2. Differentially Expressed Genes
The image of gene expression profiles in BE was shown in fig. 2,3. From the original number of 30,968 gene probes, 426 genes had a quality score of P <0 .05 and were subjected to further comparison. Of the 426 genes that had significantly different expression between BE and normal esophagus, 142 were upregulated and 284 were downregulated in BE. On the basis of Gene Ontology and KEGG Pathway, four different molecular functional pathways (cobalamin transport, cobalt ion transport, negative regulation of transcription from RNA polymerase II promoter, transition metal ion transport) were most significantly upregulated and six different molecular functional pathways (MAPK signaling pathway, T cell receptor signaling pathway, Ribosome, Wnt signaling pathway, VEGF signaling pathway, Apoptosis) were most significantly downregulated. The reverse transcription-PCR confirmed the results of microarrays. These genes were listed on table 1. These differentially expressed genes were classified based on Gene Ontology (GO) system and TreeView.
Many EAC-assoeiated genes were screened by the high-throughput gene chip method.There were 212 up-regulated genes and 126 down-regulated genes am- ong 2-fold DEGs. including 16 genes related to cytochrome P450 (CYP)
3. Confirmation by Real time RT PCR
The arrays screening results of 7 genes were conformed by real-time RT-PCR . We found that there was a good correlation between this two methods. The results of 7 target genes were not only consistent with the results of Agilent gene hybridization, but the gene expression levels were also very similar.
4. The expression of Cofilin, Intergrinβ1,RNF121 detected by TMA
The expression of Cofilin,Intergrinβ1,RNF121in BE were all significantly increased compared to normal esophagus mucous, and their expression in EAC and ESC were increased than in latero-cancer tissues. Furthermore, The expression of Cofilin,Intergrinβ1,RNF121in EAC were all significantly increased compared to in BE,but not in EAC.
5. The expression of Cofilin and RNF121 detected by Western-Blot
The expression of Cofilin and RNF121 in BE were both significantly increased compared to normal esophagus mucous, and their expression in EAC and ESC were increased than in latero-cancer tissues. Furthermore, The expression of Cofilin, RNF121in EAC were all significantly increased compared to in BE,but not in EAC. These results were matched to the results of TMA.
Conclusion
1. Two biopsies by disposable jumbo biopsy forceps provided approximately 5 microg required for microarrays. Microarray-based studies are feasible in endoscopically obtained tissues.
2. 426 different expressed genes including 142 up-regulated genes and 284 down-regulated genes were screened by gene chip. These genes involved in cell cycle, signal transduction, mucoprotein, oncogene and anti-oncogene, bone morphous protein, apoptosis inhibiting, and BCL-2 family. These different expressed genes maybe related to the genesis and development of BE, and at the same time these genes maybe related to the transformation of BE to EAC.
3. The up-regulated expression of MMP related genes、down-regulated expression of CYP related genes and gene polymorphism of CYP2 subfamily may be involved in the onset and progress of EAC.
4. The results of Real-time RT-PCR were matched to that of gene chip and both gene chip and Real-time RT-PCR were credible. Gene chip has the feature of high flux and large scale, but the Real-time RT-PCR was suit for the study of single gene. They are mutual for supplement and verification.
5. It was discovered by TMA that the expression of Cofilin ,Intergrinβ1and RNF121 were all increased in BE,EAC and ESC, and The expression of Cofilin,Intergrinβ1,RNF121in EAC were all significantly increased compared to in BE,but not in EAC. It is demonstrated that Cofilin、Intergrinβ1and RNF121 were related to the genesis of BE and played a great role for BE transformed to EAC. This was an original discovery for the mechanism of BE.
6. The results of Western-blot were matched to that of TMA. It demonstrated that TMA was a reliable and high flux protein analytical system. Gene chip combined with TMA would provide an integrity analytical system for gene expression, amplification and function detection. The results we got provided vigorous evidence for deeply understanding the genesis and improvement of BE.
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