The Expression And Significance Of Bim Protein And P-AKT In Breast Canceration

IntroductionBim is a pro-apoptotic member of the Bcl-2 protein family with only one BH3 domain,which expresses extensively in normal tissues.Bim protein plays an important role in defending autoimmune and keeping hematopoietic cell homeostasis.It also has TIF activity.Bim expression and functional regulation way are both different in different cell lines.The regulation of Bim at transcriptional level is complicated which can be regulated by kinds of signal cascades(MLK/JNK,PI3K/AKTand RAS/MAPK pathway).Many research indicated that the dysfunction of PI3K/Akt signal pathway has a close correlation with tumorigenesis and tumou development.The former research of our group indicated that PI3K-Akt-Bad pathway plays an important role in the occurrence and development of breastcancer.Bim and Bad are both belong to BH3-only family,so we are wondering if Bim plays the same role with Bad in breastcancer,which has great significance for the clinical diagnosis and therapy of breastcancer.Material and Methods1.MaterialHuman normal breast epithelium cell line MCF-10A,three human breast cancer cell lines MCF-7,MDA-MB-231及MDA-MB-435S.30 fresh breastcancer tissues and 30 adjacent breast tissues.101 breast cancer tissues staples were obtained from the First Affiliated Hospital of China Medical University from2001to2007. 2.Main reagentsRabbit polyclonal antibody Bim,p-Akt1/2/3(Ser473) and LY294002 were all purchased from Cell Signaling company.The second antibody for westernblotting,anti-β-actin抗体(PR-0255),the second antibody for fluorescence,HEST were all from Zhongshan Company.Trizol was purchased from Invitrogen company.RT-PCR kit and the primer sequence were both from TaKaRa company.3.Methods(1) Immunofluorescence was used to detect the expression of Bim in normal breast endothelial cell MCF-10A and three other breast cancer cell lines MCF-7,MDA-MB-231 MDA-MB-435S.(2) Immunohistochmistry was used to detect the expression of Bim by Immunohistochmistry S-P method.(3) Western Blotting and RT-PCR were respectively used to detect the expression of Bim protein and BimmRNA in normal breast endothelial cell and three other breast cancer cell lines.(4) Western Blotting were used to detect the expression of Bim and P-Akt in breastcancer and adjacent breast tissues.(5) Westernblotting was also used to test the relationship between the expressions of the Bim and P-Akt by inhibiting PI3K/AKT signal cascade with LY294002.(6) T test and Pearson correlation test were conducted with SPSS 13.0 statistical software to analyse Western Blotting and RT-PCR image grey scale.It has statistical significance when p＜0.05.Results1.Immunofluorescence indicated Bim protein expressed in cytoplasm of the four cell lines.2.The expression levels of Bim protein and BimmRNA were both higher in normal breast epithelium cell line than in other three breast cancer cell lines (p=0.003)(p=0.009).3.There was a statistically significant difference of Bim protein between the usual ductal hyperplasia and severe ductal atypical hyperplasia and intraductal carcinoma in situ(p=0.026).And there was statistically significant difference of Bim protein expression positive rate between the severe ductal atypical hyperplasia and intraductal carcinoma and invasive ductal carcinomas(p=0.014).The expression of Bim correlated with histological grade(p=0.033),and the lower the histological grade was,the lower the positive rate of the protein expression was.4.The Bim protein expression in adjacent breast tissues were significantly higher than that in breastcancer tissues,with lower expression of P-Akt in adjacent breast tissues(p=0.001).There was a negative correlation between the two proteins(p=0.039).5.After different LY294002(0,10,25,50μmol/L) was added into the culture fluid of MCF-7 cell line,the expression of p-Akt protein decreased while Bim protein increased(p＜0.05).Conclusion1.The expression levels of both Bim protein and Bim mRNA were higher in normal breast epithelium cell line than in other three breast cancer cell lines.2.The expression of Bim protein showed a decreasing tendency in the development of breast cancer.Bim protein had relationship with histological grade,and the lower the histological grade was,the lower the positive rate of the Bim protein expression was.3.The Bim protein expression in adjacent breast tissues were significantly higher than that in breast cancer tissues,with lower expression of p-Akt in adjacent breast tissues.There was a negative correlation between the two proteins.4.The expression of Bim protein increased after LY294002 was added which suggest the expression of Bim may be regulated through PI3K/Akt pathway.