胃癌细胞线粒体12S rRNA突变及其意义

Mutation of mitochondrial 12S rRNA in gastric carcinomas and its significance

目的检测胃癌细胞线粒体基因组(mtDNA)12SrRNA的变异,探讨其与胃癌发生的关系及意义。方法采用PCR产物直接测序法检测22例胃癌组织及其对应胃癌远端正常组织的细胞线粒体12SrRNA的变异;对其中发生特异性突变的癌组织细胞和异型增生组织细胞采用激光捕获显微切割技术进行分离,采用变性高效液相色谱法(DHPLC)、等位特异性PCR、巢式PCR和聚丙烯酰胺凝胶电泳,进一步分析该突变的性质及其突变体在癌和异型增生细胞中的定量差异;RNAdraw软件分析12SrRNA突变体RNA二级结构变化。结果(1)与mitomap线粒体数据库比较后发现一些新的变异位点,其中np652G插入和np716T G颠换仅在癌组织中。(2)12SrRNA变异的频度在弥漫型胃癌(5/17,29.4%)和肠型胃癌(12/17,70.6%)间差异有统计学意义(P<0.05)。(3)线粒体12SrRNA np652G插入和716T G颠换为异质性突变,该突变体在癌组织细胞(0.89±0.02)中的相对量高于异型增生组织细胞(0.68±0.09,P<0.01)。(4)652G的插入不利于12SrRNA局部RNA二级结构的稳定,而T G颠换等则影响有限。结论(1)12SrRNA高变异率可能与肠型胃癌的发生有关;(2)大部分变异共存于胃癌和正常胃组织中,可能不具有肿瘤特异性;(3)np652G插入和np716T G突变对胃癌发生可能有一定的作用;(4)在正常胃黏膜异型增生胃癌演?

Objective To detect the alterations of mitochondrial 12S rRNA in patients with gastric cancer, and further evaluate their effects on development of gastric carcinomas. Methods Mitochondrial 12S rRNA of 22 samples of gastric cancer tissues and 22 coresponding normal gastric mucosa taken from the distal portion of surgical specimens were PCR amplified, followed by direct DNA sequencing. Laser capture microdissection technique (LCM) was used to isolate cancerous cells and dysplastic cells from patients with specific mutations. Denaturing high-performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were applied to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplastic cells. Finally, RNAdraw bio-soft was used to analyze the RNA secondary structure of mutant type 12S rRNA. Results Compared with mitomap database, some variations were firstly found, among which np652 G insertion and np716 T-G transversion were only found in cancers. There existed statistically significant difference in variant frequency of 12S rRNA between intestinal type and diffuse type of gastric carcinoma, 5/17(29.4%)and 12/17(70.6%)respectively (P<0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutation. Variant frequency of 12S rRNA in cancer was higher than that in dysplasia(P<0.01). 12S rRNA 652G insertion had more adverse effect on secondary structure stability of 12S rRNA than T-G transversion did. Conclusion Highly variant frequency of mitochondrial 12S rRNA may be associated with intestinal type of gastric cancer. Most parts of variations exist in both cancer and normal tissues and may not be characteristic of tumor specificity. However np652 G insertion and np716 T-G transversion may possess some molecular significance on gastric cancerogenesis. During the process of progression from normality through dysplasia to cancer, 12S rRNA tended to transit from homoplasmy (wild type) and heteroplasmy to homoplasmy (mutant type, np717 T-G).