Correlation Of HMLH₁ Gene,PTEN Gene And MSI To Breast Carcinogenesis

ObjectiveBreast cancer is the common malignant tumor of women. It's incidence raises very year, it seriously threatens women's health. The pathogenesis of breast cancer is a process of multiple factors ,multiple genes and a series of steps. Studying breast cancer abnormal changes at molecular and gene level. Which have become the key point to improving the early diagnosis,early therapy and improving the prognosis of breast cancer. In our study according to detecting the protein expression of DNA mismatch repair hMLH, promotor. The state of MSI and the mutatins of PTEN in breast cancer. In order to explore the mechanism of MSI and mutations of PTEN in breast cancer from point of the methylation of mismatch repair gene promotor lend to the deticiency of mismatch repair genes result in hereditary instability trying to find a more effective genetic marker, which will make reasional foundation for research in cause of breast cancer and early diagnosis, early therapy of breast cancer.Materials and Methods1.Speimen:The fresh surgical specimen of breast cancers, totally 60 specimen, were all obtained from the First Affiliated Hospital of JIlin Uunivercity (2006-2007). After pathological diagnosis, they were kept in the refrigerator at–80℃. The cases included breast cancers and adjacent normal tissues.2.The Microsatellite instability (MSI) analysis was performed using five microsatellite markers in breast cancer by PCR.and to study the correlation with expression of hMLH₁3.We analysed the protein expressions of hMLH₁ and PTEN in 60 cases of breast cancer and 60 cases of normol breast tissues by immune- ohistochemistry S- P method. Analyze the alterations of expressions of hMLH₁,PTEN in carcinogenesis. To evaluate the correlations between expres- sions of hMLH₁,PTEN and tumorous clinical pathological characteristics.4.Promoter methylation analyses:By Methylation-Specific PCR(MSP), the hMLH₁ gene promotor is determined by chemical treatment with sodium bisulfite and stored at -20℃. The primer sequences were accordance with the literature. PCR is performed..promoter methylation analyses compare normal tissues with breast cancer tissues,analysing the relationship between methylation of hMLH₁ protmotor. and tumorous clinical pathological characteristics5.We detect the protein expressions of hMLH₁ in defferent histologic differentiation 60 cases of brenst cancer and 60 cases of normal breast tissues by western-blot ,explores the relationship between them and methylation of hMLH₁ protmotors. Western blot confirmes the immunohistochemistry resμlts.6.PTEN mutations were detected by single–strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing in breast cancer and normal breast tissues determine whether PTEN mutations is correlated with MSI and its potential mechanism.7.Statistical analysis:The Student's test. The Pearsonχ2 test or Fisher's exact probability test were used to analyze the data by SAS for Windows Release 9.0 (P-value <0.05) was considered statistically difference. Resμlts1.The respective incidence of MSI in the 5 microsatellite markers, BAT26 is 13.33%(8/60);D3S1067 is 3.33%(2/60);D2S123 is 10%(6/60);D3S1577 is 6.67%(4/60);D17S250 is 6.67%(4/60), In the 60 breast cancer analyzed 4/60(6.67%) are MSI-H,16/60(26.67%) are MSI-L,and 40/60 (66.7%) are e MSS, MSI isn't presented in normal tisssues. there is statistical difference between them,the obviously correlation was observed between MSI and differentiation.2.Expression of hMLH₁,and PTEN in breast cancer; Immunohistoche- mistry results show that hMLH₁ expression is normal or little lower in normal breast tissues; hMLH₁expression is absent or lower in breast cancer tissues with MSS; while always the main absent or lower in breast cancer tissues with MSI. PTEN expression rate is 73.33 % in normal , lower is 20 % , absent is 6.67 %; PTEN expressed usually absent (40%)or significantly lower (26.67%)in breast cancer, the main PTEN expression is absent in breast cancer with MSI.There were statistical difference between breast cancer tissues and normal tissues with hMLH₁ PTEN expression3.Methylation of hMLH₁ gene promoters: the incidence of hMLH promoter methylation is 56.67% in 60 case breast cancer, and 13.33%(8/60)in the mormal breast tissues ,there is statistical difference between them ,NO correlation is observed between the methylatim of hMLH gene promoter and differentiation lymph node invasion and tumor size,.4.Western blot confirmed the immunohistochemistry results;60cases breast cancer tissues show low expression there is statistical different between normal tissuds and breust cancer tissues5.The relationship between hMLH₁ expression and tumorous clinical pathological characteristics. The significant correlation is observed between the loss rate of hMLH₁ protein expression and differentiation, (P≤0.05) , whereas no relation was found between the loss rate and age, lymph node metastasis or tumorigenesis site of breast cancer.6.The relationship between MSI and PTEN mutations; PTEN is a substantially higher frequency of mutations in breast cancer with MSI compared with the frequency of mutations in breast cancer with MSS and very high of PTEN mutation in breast cancer tissues compared with the normal tissues .7.The relationship between PTEN expression and tumorous clinical pathological characteristics. There is no apparent correlation of PTEN expression with age,histological grade,degree of differentiation lymph node invasion and tumor size.Conclusion1.MSI maybe happen in the early time of breast cancer development,It can be a key point of early diagnosis of breast cancer.2.The expression of hMLH₁ is absent may lead to MSI happen.3.Methylation of hMLH₁ gene promoters can induce the disfunction of repair system of mismatch, methylation of hMLH₁ gene promoters may be the most important factor in occurrence of breast cancer.4.Mutuation of PTEN can induce the absence of expression of the protein. Mutation of PTEN may be the early factor in cancerization of breat cancer.5.Methylation of hMLH₁ gene promoters induce the disfunction of hMLH₁,can induce the MSI,and increase the mutation of PTEN,and induce the disfunction of the gene,this may be the pathway of development of breat cancer.