非小细胞肺癌中PTB对肿瘤抑制基因ceacam1选择性拼接的调控研究  

Study on the regulating mechanism of PTB in alternating splicing of tumor suppressor gene ceacam1 in non-small cell lung cancers

作　　者：袁榴娣[1] 丁洁[1] 缪峰[1] 

机构地区：[1]东南大学基础医学院遗传与发育生物学系,江苏南京210009

出　　处：《东南大学学报（医学版）》2006年第6期419-423,共5页Journal of Southeast University(Medical Science Edition)

摘　　要：目的:研究在非小细胞肺癌及癌旁组织中ceacam1通过选择性拼接而产生的两种转录产物的调控机制。方法:将用PCR方法获得ceacam1基因中从内含子5至外显子8长1 606 bp DNA片段插入到真核表达载体pCMV中,构建成ceacam1迷你基因模型并与ptb基因共转染,用PCR法鉴定转染后的产物变化;根据外显子7序列设计的探针GAE(16-nt)及ACE(8-nt)进行凝胶阻滞分析实验。分离与探针结合的蛋白并进行质谱分析。结果:ptb3种cDNA与ceacam1迷你基因共转染后,CEACAM lL表达水平下降,其中ptb4对迷你基因的表达产物影响最大。仅转染迷你基因的细胞中ceacam1L在两条带中所占比例为76.7%,而与ptb3种重组质粒共转染后,比例分别下降至58.3%、64.8%和54.0%。凝胶阻滞实验表明,探针GAE能与核蛋白结合,而ACE基本不能与核蛋白结合,与GAE结合的蛋白经质谱分析为PTB。结论:PTB过表达与ceacam1低表达有明显的相关性,拼接因子PTB参与ceacam1的选择性拼接。Objective To study the regulating mechanism of the different ceacaml splicing patterns between tumor tissues and their corresponding non-malignant lung tissues. Methods A 1.6 kb genomic DNA fragment containing the sequence from intron 5 to exon 8 of ceacaml gene was amplified by PCR and inserted into pCMV expression vector. The recombiant vector was co-transfected with PTBs（polypyrimidine tract binding protein） into H157 cell line . The different patterns of the two products were detected by PCR. The binding capacity was tested by gel-shift assay using the probe GAE and ACE in exon 7. The binding protein was prepared and analyzed by SELDI MASS. Results PTB-1 ,PTB-2, and PTB-4 could indeed modulate the ratio of ceacamlL and ceacamlS transcripts from the mini-gene, the amount of ceacamlL was decreased in a dose-dependent manner by over-expression of PTB. The ratio of ceacamlL in control cells was 76.7% ,it was decreased to 58.3% ,64.8% and 54.0% when co-transfected with three isoforms of PTB. The GAE probe could strongly bind with nuclear protein, while the 8-nt ACE with a single CACA element could not. The binding protein with GAE was proven to be PTB by mass spectrometry. Conclusion Over-expression of PTB is correlated with the alternative processing of ceacam1L. PTB is the splicing factor that involves in inclusion/exclusion of ceacaml exon 7.

关 键 词：迷你基因 选择性拼接 癌胚抗原相关的细胞粘附分子1 拼接因子PTB 凝胶阻滞分析 非小细胞肺癌 

分 类 号：Q753[生物学—分子生物学] Q756