一氧化氮诱导食管癌细胞线粒体DNA编码基因过表达

Overexpression of Mitochondria DNA Encoded Genes in Human Esophageal Carcinoma Cell Line Induced by Nitric Oxide

以人食管癌细胞系EC10 9作为驱赶方 (driver) ,以被一氧化氮 (nitricoxid ,NO)诱导的EC10 9作为实验方 (tester) ,应用抑制消减杂交 (suppressionsubtractivehybridization ,SSH)、反向mRNA斑点印迹和RNA印迹等技术手段研究了NO诱导的食管癌细胞中基因的过表达情况 .然后对过表达基因的表达序列标签 (expressedsequencetag ,EST)实施序列测定 ,并与GenBank进行BLAST同源性比较和序列突变分析 .结果先后两次从 6 9个SSH阳性克隆中共鉴定出 6个线粒体DNA (mitochondrialDNA ,mtDNA)编码的基因 ,即ND 4L、ND 4、COX 2、Lys tRNA、ATP 8和ATP 6 .表明NO可以诱导食管癌细胞mtDNA编码的基因过表达 .另外 ,在ND 4L/ND 4基因的片段 (10 736～ 114 4 9)上发现了三处同型单核苷酸置换 (10 872T→C ,110 0 1A→G ,11346A→G) ,在COX 2 /Lys tRNA/ATP 8/ATP 6基因片段 (80 11～ 85 89)上发现了一处单核苷酸缺失(8380A) .氨基酸序列分析表明 ,在NO诱导的EC10 9中可能存在着一种结构异常的ATP 8肽链 (一条在N端被截短的只有 11个氨基酸残基的肽链 ,而正常的ATP 8肽链为 6 8个氨基酸残基 ) .

Overexpression of the genes induced by nitric oxide (NO) in EC109 esophageal carcinoma cell line was studied by using suppression subtractive hybridization (SSH), reverse mRNA dot blot and Northern blot. The nucleotide of their expressed sequence tag (EST) was sequenced and analyzed by NCBI database. The six mitochondrial DNA coding genes, ND-4L, ND-4, COX-2, Lys-tRNA, ATP-8 and ATP-6 were identified from 69 SSH positive clones. The results indicated that NO can distinctly induce overexpression of mitochondria DNA encoded genes in the esophageal carcinoma cells. In addition, it was discovered that there were single nucleotide substitution in three sites of the fragment of ND-4L and ND-4 genes (10 736similar to11 449, 10 872 T-->C, I 1 001 A-G, 11 346 A-G), and one single nucleotide deletion (8 380, A) which will lead to occur the frame-shift mutation in the peptide in the fragment of COX-2/Lys-tRNA/ATP-8/ATP-6 genes (8011similar to8589). The analysis of amino acid sequences showed that an incorrect structural ATP-8 subunit existed possibly in the EC109 cell induced by NO. These results provided a new clue for further exploring the mechanism of NO effecting on carcinoma cells.