Study Of The Nteraction Between Microsatellite Instability And Mutation Of Mismatch Repair Genes In Breast Cancer

Objective:Breast cancer is the common malignant tumor of women.The gene of mammary gland cellula epithelialis became mutate for the multitude carcinogenic agents,it induce to the cellular proliferation loss of control and make breast cancer happened.The pathogenesis of breast cancer is a process of multiple factors,multiple genes and a series of steps.But the hereditary instability is the most important genetics effect of the pathogenesis of breast cancer,the hereditary instability have two different types:one is microsatellite instability(MSI) and the other one is loss of heterozygosity(LOH).The mutation of mismatch repair genes(MMR),especially the tow important hMSH2 and hMLH1 have closs relationship wite MSI,it will lend to the deficiency of mismatch repair genes result in hereditary instability and cause the MSI happened.Studying breast cancer abnormal changes at molecular and gene level, Which have become the key point to improve the early diagnosis and accurate predict prognosis of breast cancer.In our study in order to explore the mechanism of MSI and mutations of hMSH2 in breast cancer,we detecting the protein expression of DNA mismatch repair hMSH2,the mutation of its exon and the state of MSI in breast cancer.Trying to find a more effective genetic marker, which will make reasonable foundation for research in the cause of breast cancer and prognosis predict of breast cancer.Methods:78 sample of breast cancers,39 sample of benign tumor of breast and 18 sample of breast tissues away from cancer as control were studied. All samples were obtained from the Affiliated Tumour Hospital of Guangxi Medical University between 2004 and 2008.The protein expression of hMSH2 were detected by immuneohistochemistry S-P method.DNA extracted by DNA extracted kit.The primer sequences of hMSH2 exon were design using the oligo 6 software.Fragments of all exons were amplified by plymerase chain reaction(PCR),and then hMSH2 exons mutations were detected by single-strand conformation polymorphism analysis(PCR-SSCP).Sequencing the DNA which the results of SSCP were abnomal.The five microsatellite situs were choosed according to the literature and findout their sequences in gene bank.The MSI were analyzed by PCR-single strand length polymorphism(SSLP).Results were statistically analyzed by SPSS13.0 software.Categorical data using chi-square test(χ2 test),ordinal data using rank sum test,multivariate analysis using logistic regression.A p value less than 0.05 was considered significant.Result①Immunohistochemistry results show that hMSH2 expression rate in 18 breast tissues away from cancer,39 breast benign tumor tissues and 78 breast cancer tissues is 94.44%,76.92%and 39.74%respectively.There were statistical difference in these three group(χ2=29.383,p=0.000).The expression of hMSH2 in breast cancer tissues is decrease obviously.②The significant correlation is observed between the decrease rate of hMSH2 protein expression and tumour sise,clinical stage and axillary nodes metabasis.The decrease rate of hMSH2 protein expression is more easily observed in the tissues with big tumour sise,later clinical stage and have axillary nodes metabasis.③It was no hMSH2 gene mutation in the breast tissues away from cancer.1 mutation in exon 7(409C→G) was found in these 39 breast benign tumor tissues.There totally 9 mutation in breast cancer tissues,including 2 mutation in exon 6 (322G→A),1 mutation in exon 7(378A→T),1 mutation in exon 12(623C→G),1 samesense mutation in exon 12(631A→G),1 mutation in exon 13(727T→G),2 samesense mutation in exon 14(796A→C) and 1 mutation in exonl4(801A→G).The rate of mutation in breast cancer tissues is 11.54%. There were statistical difference in these three group(χ2=4.332, p=0.037).hMSH2 mutation was more easily observed in the breast cancer tissues.④MSI isn't presented in breast tissues away from cancer,5 of 39 breast benign tumor tissues can found MSI in at least one microsatellite situs,the rate of MSI is 12.82%.35 of 78 breast cancer tissues can found MSI in at least one microsatellite situs,the rate of MSI is 44.87%.There were statistical difference in these three group(χ2=20.365,p＜0.05),MSI was more easily observed in the breast cancer tissues.⑤The significant correlation is observed between the MSI and these tumour sise,clinical stage and axillary nodes metabasis of the breast cancer.The MSI is more easily observed in the tissues with later clinical stage and have axillary nodes metabasis.⑥The hMSH2 mutation is significant correlation with decrease of hMSH2 protein expression (χ2=6.624,p=0.01) and MSI(χ2=10.572,p=0.01).In breast cancer,all hMSH2 mutaton samples can observed the decrease of hMSH2 protein expression and MSI.Conclusion①hMSH2 protein is high expression in breast tissues away from cancer.hMSH2 protein expression is decrease in breast benign tumor tissues,in breast cancer tissues it decrease the most obviously.②The decrease rate of hMSH2 protein expression is more easily observed in the breast cancer tissues with big tumour sise,later clinical stage and have axillary nodes metabasis,indicate that hMSH2 canbe a genetic marker for predicted prognostic of breast cancer.③hMSH2 have no mutational hot spot in breast cancer, mutation of hMSH2 is distribute in exon 6,7,12,13 and 14.In breast cancer hMSH2 mutation may be a mechanism of the disfunction of MMR system.④MSI is widespread in breast cancer,it is more easily observed in the tissues with later clinical stage and have axillary nodes metabasis,indicate that MSI canbe a genetic marker for clinical stage of breast cancer,it helps to predicted prognostic of breast cancer.Patient with MSI maybe poor prognostic.