The Distribution And The Expression Of BAMBI In Breast Cancer Cell Lines

IntroductionBreast cancer is the most common human cancer and is the primary factor in cancer death worldwide.Therefore,as the strengthening of breast cancer in molecular biology and other fields as the basis for the etiology of breast cancer,exploring its pathogenesis and treatment is extremely important.BAMBI(BMP and activin membrane-bound inhibitor)gene in chromosome 10p12.3-p11.2 region,coding for a product by the 260 amino acid composition of the transmembrane glycoprotein,molecular weight of 29 108 Dalton,which also known as fake receptor(pseudoreceptor),NMA(non-metastatic A protein gene),belonging to the BAMBI family.The family members have similar extracellular domain structure with type I receptor ligand,such as TGF-β(the transforming growth factor-β,TGF-β), Bone morphogenetic protein(bone morphogenetic protein,BMP),activin.Therefore BAMBI can integration of ligand-receptor complexes,and form TGF-forming polymer with TGF-βⅡreceptor.But since it does not have TGF-typeâ… receptor-specific serine/threonine kinase domain.It can not phosphorylated Smad located in cytoplasm of protein.,thereby blocking receptor of TGF-signal transduction.It is reported BAMBI were up-regulated expression of existence in lung colon carcinoma,liver cancer and whether express in breast cancinoma yet not clearly.ObjectiveTo study the expression and its significance of BAMBI in breast cancer and explore the relation beween BAMBI and clinical and pathological factors of breast cancer. Materials and Methods1.Breast Cancer Cell LinesThree Breast cancer cell lines including MDA-MB-435s,MDA-MB-231,MCF-7 were used for immunofluorescence technology,RT-PCR and Western blot.2.Semi-quantitative RT-PCRRNAout used to extract the total RNA,synthesis of BAMBI and primer of GAPDH.To synthesis Cdna chain by two-step RT-PCR kit,then amplification.The gel electrophoresis imaging analysis system were used to image acquisition of production by 2%agarose gel electrophores,then we calculated the integral optical density value of the band.3.Cell CultureMDA-MB-435s,MDA-MB-231,MCF-7 cells were cultured in the presence of 10%fetal calf serum DMEM.The conditions of incubation are 37℃,5%CO₂.4.Western blot ProtocolCentrifugal collection of cells adding some lysate,take the supernatant.After SDS-PAGE electrophoresis,transfer the membrane,incubation in primary antibody (1:1000)-4℃overnight after blockage.Incubation in HRP-conjugated secondary antibody(1:10000)for 2h at room temperature next day,using ECL reagent in darkroom and exposure,Gel electrophoresis imaging analysis system for capturing the image and calculating integral optical density value of the bands.5.Immunofluorescence ProtocolThe coverglass-cultured cell were fixed by formalin,incubation in primary antibody(1:200),-4℃overnight after blockage.Incubation in FITC-conjugated secondary antibody(1:100)for 2h at room temperature next day,nuclear reataining with PI(1:1000),mounting.The immunofluorescence were observed and imaged by the laser scanning confocal microscope image acquisition system. ResultsBAMBI protein expressed mainly in the membrane and the cytoplasm close to the membrane.BAMBI mRNA expression in the MDA-MB-435s was higher in MDA-MB-231 and its expression in the MDA-MB-231 was higher than MCF-7. Respectively,the difference was significant(P＜0.05).The relative amount of BAMBI protein in MDA-MB-435s,MDA-MB-231 and MCF-7 were 0.963±0.061, 0.957±0.048,0.769±0.103,and the difference was significant(P＜0.05).ConclusionThe result of experiments has revealed that the abnormally high expression of BAMBI has relationship to breast cancinogenesis.The expression of BAMBI has relationship with the metastasis of breast cancer.