大肠癌组织CEA基因启动子序列克隆及与正常组织相应序列的比较

Cloning and sequencing analysis of carcinoembryonic antigen promoter from freshly isolated human colorectal carcinoma with relative DNA fragment from normal tissues

目的：研究人类不同组织细胞中癌胚抗原（ＣＥＡ）产生与其基因顺式作用序列改变的关系，评价该顺式作用序列在大肠癌组织特异性基因治疗中的应用价值。方法：采用套式ＰＣＲ方法分别从人新鲜大肠癌、相应癌旁粘膜组织、正常血细胞、正常胎盘组织及建株癌基因组中扩增ＣＥＡ基因启动子核心序列，ＰＣＲ－Ｓｏｕｔｈｅｒｎ杂交法确定扩增序列可靠性。用ＰＣＲ－单链构象多态性（ＳＳＣＰ）和ＤＮＡ序列分析方法确定扩增序列突变情况。用Ｂａｎｄ－Ｓｈｉｆｔ分析法检验扩增序列的启动子功能。结果：不同组织细胞基因组中的ＣＥＡ基因转录起始位点上游－１３０ｂｐ～＋６９ｂｐ的ＣＥＡ基因启动子核心序列区段未发现明显序列差异。大肠癌组织中ＣＥＡ基因的转录与大肠癌基因组中ＣＥＡ基因启动子核心序列无明显的对应关系。从大肠癌组织和正常组织ＤＮＡ中扩增出来的ＣＥＡ启动子序列与ＣＥＡ阳性的ＬｏＶｏ大肠癌细胞核蛋白提取物的结合能力一致，但不能与ＣＥＡ阴性的ＲＣ９４０６人肾癌细胞结合。结论：ＣＥＡ基因在大肠癌细胞中的特异转录不是由于ＣＥＡ基因启动子序列的突变。来源于正常细胞或者大肠癌细胞基因组的ＣＥＡ基因启动子均适合大肠癌组织特异性基因治疗。

Objective: To investigate the relationship between production of carcinoembryonic antigen (CEA) in the human cells of different tissues and sequence variation of CEA promoter in the cellular genomes, and evaluate potential usage of the cis active sequence in the colorectal carcinoma tissue specific gene therapy. Methods: Nest polymerase chain reaction (PCR) was employed to amplify the CEA gene promoter sequences from the genomes of freshly isolated colorectal carcinomas, normal adjacent colonic mucosa, normal blood cells, placenta tissue and some established cell lines of human origin. PCR Southern bloting was utilized to define the reliability of the amplified sequences. Sequence variation was screened by means of PCR single stranded conformation polymorphism (SSCP) and DNA sequencing. Results: No sequence variation was found between -135 bp～ + 69 bp from the transcriptional initiation site, which was considered to be a core region of the CEA gene. There was no correlativity between expression levels of CEA gene and sequence variations of CEA gene promoter core region. The fragment amplified either from normal tissues or from colorectal carcinoma tissues equally bound with the nuclear extract from CEA positive LoVo human colorectal carcinoma cells, and could not bind with the extract from CEA negative RC9406 human renal carcinoma cells. Conclusion: Specific transcription of CEA gene in colorectal carcinoma cells is inferred not to be caused by the variation of cis active sequence in the genome of colorectal carcinoma cells. CEA promoter sequence from normal human genome is suitable for colorectal carcinoma tissue specific gene therapy.