Mutations of mitochondrial 12S rRNA in gastric carcinoma and their significance

Abstract

Aim: To detect the variations of mitochondrial 12S rRNA in patients with gastric carcinoma, and to study their significance and the relationship between these variations and the genesis of gastric carcinoma.

Methods: PCR amplified mitochondrial 12S rRNA of 44 samples including 22 from gastric carcinoma tissues and 22 from adjacent normal tissues, was detected by direct DNA sequencing. Then laser capture microdissection technique (LCM) was used to separate the cancerous cells and dysplasia cells with specific mutations. Denaturing high performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw biosoft was used to analyze the RNA secondary structure of mutant-type 12S rRNA.

Results: Compared with Mitomap database, some new variations were found, among which np652 G insertion and np716 T-G transversion were found only in cancerous tissues. There was a statistic difference in the frequency of 12S rRNA variation between intestinal type (12/17, 70.59%) and diffusive type (5/17, 29.41%) of gastric carcinoma (P<0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutations. The frequency of 12S rRNA variation in cancerous cells was higher than that in dysplasia cells (P<0.01). 12S rRNA np652 G insertion showed obviously negative effects on the stability of 12S rRNA secondary structure, while others such as T-G transversion did not.

Conclusion: The mutations of mitochondrial 12S rRNA may be associated with the occurrence of intestinal-type gastric carcinoma. Most variations exist both in gastric carcinomas and in normal tissues, and they might not be the characteristics of tumors. However, np652 G insertion and np716 T-G transversion may possess some molecular significance in gastric carcinogenesis. During the process from normality to dysplasia, then to carcinoma, 12S rRNA tends to convert from homoplasmy (wild type) to heteroplasmy, then to homoplasmy (mutant type, np717 T-G).

Figure 1

Sequencing results of mitochondrial 12S rRNA in gastric cancer. A: Normal 12S rRNA (652G); B: 12S rRNA 652G insertion ; C: Normal 12S rRNA (716T, 728C); D: 12S rRNA 716 T-G transversion , 728 C-T transition.

Figure 2

Analytic results obtained by denaturing high performance liquid chromatography. A: np 652G insertion, heteroplasmic mutation; B: np 716 T-G transversion, heteroplasmic mutation.

Figure 3

Mutant property of 12S rRNA 716T-G shown in AS-PCR analysis. A: gastric cancerous cells; B: dysplasia cells. Lanes 1-5: mutant type 12S rRNA; lane 6: pGEM-7ZF marker (HuaMei) and 100 bp Ladder marker (TaKaRa) respectively; lanes 7-11: wild type 12S rRNA; lanes 12, 13: mix type 12S rRNA of gastric cancerous cells and dysplasia cells respectively.

Figure 4

RNA secondary structure prediction of mutant type of 12S rRNA. dG: free enegy of RNA secondary structure (kcal/mol); ins: insertion. Arrowhead indicates local RNA structural change.