食管癌Mal基因5′调控区的克隆及序列分析

The cloning and sequence analysis of 5′regulatory region of Mal gene of esophageal cancer

目的 探讨Mal基因在人食管癌中表达下调的机制。方法 Mal基因 5′调控区是一高G+C含量的区域 ,总G +C含量占 6 7.0 8% (4 87/ 72 6个 ) ,经优化PCR反应条件扩增正常胎盘组织和人食管癌细胞系EC970 6、EC8712、EC10 9的Mal基因 5′端调控区 ,对PCR产物进行克隆和序列测定 ,对测序结果进行相互比较。结果 成功地扩增出Mal基因 5′调控区。与正常胎盘组织相比 ,食管癌细胞系EC970 6、EC8712、EC10 9均有Mal基因 5′调控区第 6 5 4位的T→C改变 ,相应的密码子TCC变为CCC ,所编码的氨基酸由丝氨酸变为脯氨酸 ;EC970 6还有第 6 5 7位的T→C改变 ,相应的密码子TGC变为CGC ,所编码的氨基酸由半胱氨酸变为精氨酸 ;EC8712有第 139位的A→G改变 ,相应的密码子GGA变为GGG ,所编码的甘氨酸仍为甘氨酸。结论 建立了Mal基因 5′调控区的PCR扩增方法 ;食管癌Mal基因 5′调控区可能存在点突变。

Objective To investigate the mechanisms of Mal gene down regulated in human esophageal cancer. Methods We amplified the 5′regulatory region of Mal gene in normal placenta tissue and human esophageal cancer cell lines EC9706、EC8712、EC109 by optimization the PCR methods, cloned and sequenced the PCR products and compare the sequence. Results Human esophageal cancer cell lines EC9706、EC8712、EC109 all have the 654 th of 5′regulatory region of Mal gene T to C changes, corresponding codon changed from TCC to CCC, and the amino acid which it codes changed from serine to proline; EC9706 also has the 657 th of 5′regulatory region of Mal gene T to C changes, corresponding codon changed from TGC to CGC, the amino acid which it codes changed from cysteine to arginine; EC8712 has the 139 th of 5′regulatory region of Mal gene A to G changed, corresponding codon changed from GGA to GGG, the amino acid which it codes remains glycine. Conclusion Our results suggested there are may point mutation in 5′ region of Mal gene in human esophageal cancer.