Study On Microsatellite Instability And HMLH1 Promoter Methylation In Gastric Cancers And Precancerous Lesions

PurposeTo study the relationship among MSI, the expression of hMLH1 and methylation of the hMLH1 promoter , the frameshift mutation of TGF-β R â…¡ in (A)₁0 tract in gastric cancer and precancerous lesions, and to explore their action in gastric carcinogenesis and the molecular mechanism in cancer development. We hope that it can give help on early diagnosis prophylaxis and treatment of gastric cancer. Methods101 cases were studied, including 41 cancers, 46 precancerous lesions (20 intestinal metaplasia adjacent to cancers, chronic atrophic gastritis with intestinal metaplasia and atypical hyperplasia without cancers) and 14 normal gastric mucosa. The correspondent normal tissue was used as the control. DNA was isolated by phenol-chloroform method, microsatellite loci D2S123 and BAT-26 were used to detect MSI status. PCR was performed and then the products were electrophoresed on denatured polyacrylamide gel .Silver staining was used to decide the result, the frameshift mutation of TGF- 3 RII was also screened with the same method. The methylation of hMLH1 promoter was analyzed by restriction endonuclease and specific PCR. The expression of hMLH1 wasdetected with immunohistochemistry. Results:l.The incidence of MSI was 51.2%(12/40), 26.1%(12/46), 0 in cancer, precancerous lesions and normal groups respectively. The frequency of MSI was far more higher in gastric cancer than the other two groups(P<0.05),and the frequency of RER+ was significantly higher in intestinal type than diffuse type carcinomas(P<0.05). The frequency of RER+ was significantly higher in precancerous lesions with cancers than those without cancers(P<0.05).2. Each locus of MSI in tumor was higher than non-neoplasia, and the incidence in dinucleotide microsatellite sequence was more frequent than mononucleotide in every groups.3. The frequency of hMLH1 promoter methylation in gastric cancer was 41.5%( 17/41), premalignant cases was 19.6%(9/46),and none was found in normal group. (2)The frequency of methylation was higher in cancer compared with the other two groups exhibiting MSI (P<0.001). All of the MSI-H cases were methylatedl00%(14/14), the frequency of methylation in MSI-L was 57.9%(11/19), and only one of the MSS was hypermethylated(1.5%). The frequency of methylation was higher in MSI-positive cases than MSS. 88.5% cases exhibiting hypermethylation displayed aberrant hMLH1 expression.4. The frequency of frameshift mutation in (A)10 tract of TGF- P RII with MSI-H was 35.7%(five of fourteen ), but no mutation was found in MSI-L and MSS cases. We found that the mutation was significant higher (P<0.05) in MSI-H than MSI-L and MSS.Conclusion:Our study shows that MSI takes effect on carcinogenesis and development of sporadic gastric cancer. Methylation of hMLH1 promoter is the important factor which results in inactivation of hMLH1 gene, thus lead to MSI and mutation of tumor suppress gene, and accelerate the development of tumor. Studying the conditions of MSI in tissue can be very important in the early diagnosis and/or prophylaxis of human tumors.