直肠癌相关基因的生物信息学分析及实验室初步验证
摘要
目的：结直肠腺癌(colorectal adenocarcinoma，CRA)是我国最常见的恶性肿瘤之一，发病率高，占消化道恶性肿瘤的第二位，且呈逐渐增高的趋势。目前的研究认为从正常结直肠上皮细胞到结直肠腺癌细胞的演变是多步骤、多基因逐步发展的结果，其发生发展过程与多个基因的时空异常表达以及蛋白质的相互作用有关。因此研究结直肠腺癌相关基因的表达，对研究结直肠腺癌病变分子机理具有重要意义。
从抑制性消减杂交结合cDNA芯片技术筛选发现的差异表达的表达序列标签(expression sequence tag，EST)中，选择四个EST片段，基因登录号分别为ES274071、ES274070、ES274084、ES274075。为进一步研究其与结直肠腺癌的发生发展的关系，本实验对其所代表的基因进行一系列的生物信息学分析并实验室初步验证。
方法：本实验中，首先将ES274071这个EST片段(420bp)作为种子序列，通过生物信息学的方法，利用Genbank数据库，电子克隆并通过DNASTAR软件拼接得到这一个基因的全长cDNA序列，并对其进行开放读码框(open reading frame，ORF)的预测、染色体定位、基因的组织表达谱分析，及其所编码的蛋白质同源性分析，二级结构及其功能的预测等。然后利用RT-PCR方法对拼接的片段进行了实验验证，并将ES274071的测序结果提交至Genbank，基因登录号为EF534308。对另一个EST片段ES274070，本实验也对其所代表的基因序列进行了ORF的实验室验证。对ES274084和ES274075进行了生物信息学分析。
结果：通过网上电子克隆并利用DNASTAR软件拼接了ES274071的全长cDNA序列，长度为2790bp，比原来的EST片段(420bp)在5’端延伸了1550 bp，3’端延伸了820bp。其定位于人类染色体的9q34，ORF预测为834bp并通过测序加以验证，其编码蛋白质为SETtranslocation，其中在29-225位有一个核小体组装蛋白(Nucleosome AssemblyProtein，NAP)区域。同时在NCBI网站的Unigene数据库里发现有一已知序列与其同源，基因登陆号为NM003011，长度为2577bp，而本实验通过拼接得到的全长序列较这个已登陆的序列在5’端延伸了305 bp。另一个EST即ES274070所到表基因的ORF为336bp，利用DNA测序技术加以验证。ES274084和ES274075经分析实际为同一序列，其所代表基因的编码蛋白可能为Ⅰ型胶原蛋白。
结论：将组装的ES274071的cDNA的ORF序列所编码的蛋白进行blastp检索发现其编码蛋白为SETtranslocatio。SET是肿瘤抑制因子NM23(Non-metastatic gene)及蛋白质磷酸脂酶2A(protein phosphatase2A，PP2A)的抑制剂。PP2A是一种磷酸化酶，属于抑癌蛋白，过度表达能够导致细胞周期障碍，它可调节细胞的增殖、生长及分化，而且在caspases介导的细胞凋亡中也起作用。NM23属抑癌基因，可以抑制细胞活性，抑制原位肿瘤的发生和转移，它的低表达在大肠癌的浸润和组织分化程度中起重要作用。SET含有一个ATP非依赖的核小体装配蛋白(Nucleosome Assembly Protein，NAP)的结构域，SET的NAP活性增强了染色质的可亲和性。
Objective: Colorectal adenocarcinoma(CRA) is one of the most comman malignant cancer in our country.It has a high disease incidence,account for the second status of malignant cancer in alimentary canal,and the incidence is growing year by year. The present studies indicated there are multi-gene and multi-stage mutations steps in the process of occurrence and development from normal colorectal epithelial cell to carcinomatous cell.It has been happened that there are abnormal cell division in differentiation.There are also some interactionamong proteins.So it is very important to study genes differential expression in the CRA.Furthermore,the study will support some references to explore molecular mechanism of CRA.
Filtering through suppression subtractive hybrization and cDNA chip technique ,we select four EST segments that the expression is different between colorectal adenocarcinoma tissue and normal colorectal tissue on gene chips, we named them separately as follows: ES274071,ES274070,ES274084,ES274075.In order to study the relationships furtherly with CRA,this experiment take some bioinformatics analysis and preliminary experiments to validate,so as to offer some informations for studying etiopathogenesis of CRA.
Methods:In this experiment,we selected the EST of ES274071 as a seeded sequence ,utilized the method of bioinformatics ,took use of Genbank database to clone full-length cDNA from the EST fragment.It was predicted the ORF,chromosome location,tissue distribution,genetic orgenization expression pedigree,protein homology,protein's secondary structure and function.Then the experiment of RT-PCR was employed to validate the contig.Then the sequencing result was submitted Genbank and acquired accession number that is EF534308.We also took some validation experiment about ES274070's ORF.As ES274084 and ES274075,we took some bioinformatics analysis.
Results:By electronic clone and DNASTAR software ,we assemble the matching sequence together to form a 2790bp cDNA sequence,compared to original EST sequence(ES274071,420bp),this sequence extend 1550bp from 5'end and 820bp from 3'end.It's located in human chromosome9q34,ORF's length is 834bp and has validated through experiment.It's coded protein is SETtranslocation,and there is a NAP domain during 29-225bp.In the same time,we found there is also a registered sequence which is homologous with the assemblely sequencers accession number is NM003011,the length is 2577bp.In this experiment,we assembled the full-length cDNA extend 309bp compaired with NM003011.Another EST(ES274070)represented gene's ORF is 336bp length by DNA sequencing technology.The other two EST(ES274084 and ES274075),through analysis ,we found in fact they are the same sequencers coded protein maybe human collagen protein,type I .
Conclusion:We retrieve blastp with assembled cDNA's ORF of ES274071,we found its coded protein is SETtranslocatio.SET is a suppressor of PP2Aand NM23.PP2A is one kind of phosphorylase, it is a tumor suppressor protein, its over-expression could lead to disorder of cell cycle,it contributes to cell proliferation ,growth and division, and also has some usefulness during the cell apoptosis mediated by caspases.NM23 pertaining to anti-oncogene,can supress the cell activity, the genesis and metabasis of tumor in situ,its lower expression plays an important role during the infiltrationin and metabasis in cancer of colon. SET has a non-dependent nuclesome assembly protein,its NAP activity can enhance the affinity of chromatin.
从抑制性消减杂交结合cDNA芯片技术筛选发现的差异表达的表达序列标签(expression sequence tag，EST)中，选择四个EST片段，基因登录号分别为ES274071、ES274070、ES274084、ES274075。为进一步研究其与结直肠腺癌的发生发展的关系，本实验对其所代表的基因进行一系列的生物信息学分析并实验室初步验证。
方法：本实验中，首先将ES274071这个EST片段(420bp)作为种子序列，通过生物信息学的方法，利用Genbank数据库，电子克隆并通过DNASTAR软件拼接得到这一个基因的全长cDNA序列，并对其进行开放读码框(open reading frame，ORF)的预测、染色体定位、基因的组织表达谱分析，及其所编码的蛋白质同源性分析，二级结构及其功能的预测等。然后利用RT-PCR方法对拼接的片段进行了实验验证，并将ES274071的测序结果提交至Genbank，基因登录号为EF534308。对另一个EST片段ES274070，本实验也对其所代表的基因序列进行了ORF的实验室验证。对ES274084和ES274075进行了生物信息学分析。
结果：通过网上电子克隆并利用DNASTAR软件拼接了ES274071的全长cDNA序列，长度为2790bp，比原来的EST片段(420bp)在5’端延伸了1550 bp，3’端延伸了820bp。其定位于人类染色体的9q34，ORF预测为834bp并通过测序加以验证，其编码蛋白质为SETtranslocation，其中在29-225位有一个核小体组装蛋白(Nucleosome AssemblyProtein，NAP)区域。同时在NCBI网站的Unigene数据库里发现有一已知序列与其同源，基因登陆号为NM003011，长度为2577bp，而本实验通过拼接得到的全长序列较这个已登陆的序列在5’端延伸了305 bp。另一个EST即ES274070所到表基因的ORF为336bp，利用DNA测序技术加以验证。ES274084和ES274075经分析实际为同一序列，其所代表基因的编码蛋白可能为Ⅰ型胶原蛋白。
结论：将组装的ES274071的cDNA的ORF序列所编码的蛋白进行blastp检索发现其编码蛋白为SETtranslocatio。SET是肿瘤抑制因子NM23(Non-metastatic gene)及蛋白质磷酸脂酶2A(protein phosphatase2A，PP2A)的抑制剂。PP2A是一种磷酸化酶，属于抑癌蛋白，过度表达能够导致细胞周期障碍，它可调节细胞的增殖、生长及分化，而且在caspases介导的细胞凋亡中也起作用。NM23属抑癌基因，可以抑制细胞活性，抑制原位肿瘤的发生和转移，它的低表达在大肠癌的浸润和组织分化程度中起重要作用。SET含有一个ATP非依赖的核小体装配蛋白(Nucleosome Assembly Protein，NAP)的结构域，SET的NAP活性增强了染色质的可亲和性。
Objective: Colorectal adenocarcinoma(CRA) is one of the most comman malignant cancer in our country.It has a high disease incidence,account for the second status of malignant cancer in alimentary canal,and the incidence is growing year by year. The present studies indicated there are multi-gene and multi-stage mutations steps in the process of occurrence and development from normal colorectal epithelial cell to carcinomatous cell.It has been happened that there are abnormal cell division in differentiation.There are also some interactionamong proteins.So it is very important to study genes differential expression in the CRA.Furthermore,the study will support some references to explore molecular mechanism of CRA.
Filtering through suppression subtractive hybrization and cDNA chip technique ,we select four EST segments that the expression is different between colorectal adenocarcinoma tissue and normal colorectal tissue on gene chips, we named them separately as follows: ES274071,ES274070,ES274084,ES274075.In order to study the relationships furtherly with CRA,this experiment take some bioinformatics analysis and preliminary experiments to validate,so as to offer some informations for studying etiopathogenesis of CRA.
Methods:In this experiment,we selected the EST of ES274071 as a seeded sequence ,utilized the method of bioinformatics ,took use of Genbank database to clone full-length cDNA from the EST fragment.It was predicted the ORF,chromosome location,tissue distribution,genetic orgenization expression pedigree,protein homology,protein's secondary structure and function.Then the experiment of RT-PCR was employed to validate the contig.Then the sequencing result was submitted Genbank and acquired accession number that is EF534308.We also took some validation experiment about ES274070's ORF.As ES274084 and ES274075,we took some bioinformatics analysis.
Results:By electronic clone and DNASTAR software ,we assemble the matching sequence together to form a 2790bp cDNA sequence,compared to original EST sequence(ES274071,420bp),this sequence extend 1550bp from 5'end and 820bp from 3'end.It's located in human chromosome9q34,ORF's length is 834bp and has validated through experiment.It's coded protein is SETtranslocation,and there is a NAP domain during 29-225bp.In the same time,we found there is also a registered sequence which is homologous with the assemblely sequencers accession number is NM003011,the length is 2577bp.In this experiment,we assembled the full-length cDNA extend 309bp compaired with NM003011.Another EST(ES274070)represented gene's ORF is 336bp length by DNA sequencing technology.The other two EST(ES274084 and ES274075),through analysis ,we found in fact they are the same sequencers coded protein maybe human collagen protein,type I .
Conclusion:We retrieve blastp with assembled cDNA's ORF of ES274071,we found its coded protein is SETtranslocatio.SET is a suppressor of PP2Aand NM23.PP2A is one kind of phosphorylase, it is a tumor suppressor protein, its over-expression could lead to disorder of cell cycle,it contributes to cell proliferation ,growth and division, and also has some usefulness during the cell apoptosis mediated by caspases.NM23 pertaining to anti-oncogene,can supress the cell activity, the genesis and metabasis of tumor in situ,its lower expression plays an important role during the infiltrationin and metabasis in cancer of colon. SET has a non-dependent nuclesome assembly protein,its NAP activity can enhance the affinity of chromatin.
引文
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