The Expression Of BAMBI In Non-small Cell Lung Cancer

IntroductionLung cancer is the most common human cancer and is the primary factor in cancerdeath worldwide. It has been reported that the incidence of lung cancer in Chinaincrease 26.9% each year. Therefore, the strengthening of lung cancer in molecularbiology and other fields as the basis for the etiology of lung cancer, explore itspathogenesis 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 thetransmembrane glycoprotein, molecular weight of 29 108 Dalton, which also known asfake receptor (pseudoreceptor), NMA (non-metastatic A protein gene), belonging tothe BAMBI family. The family members have similar extracellular domain structurewith typeâ… receptor ligand, such as TGF-β(the transforming growth factor-β, TGF-β),Bone morphogenetic protein (bone morphogenetic protein, BMP), activin. ThereforeBAMBI can integration of ligand-receptor complexes, and form TGF-forming polymerwith TGF-βⅡreceptor. But since it does not have TGF-typeâ… receptor-specificserine/threonine kinase domain. It can not phosphorylated Smad located in cytoplasmof protein, thereby blocking receptor of TGF-signal transduction. Studies show that tolung epithelial tissue tumor, TGF-βcan inhibit the proliferation of tumor cells, BAMBIand TGF-βsignaling pathway in the mechanism of negative regulation of lung cancer isunclear. Also, it is reported BAMBI were up-regulated expression of existence inhuman colon carcinoma, liver cancer and whether express in non-small cell lungcarcinoma yet clearly.ObjectiveTo study the expression and its significance of BAMBI in non-small cell lung cancer(NSCLC) and explore the relation between BAMBI and clinical and pathologicalfactors of NSCLC. To discuss the relation between the expression of BAMBI andNSCLC pathological type and invasion potentiality by its distribution in NSCLC cellline.Materials and Methods1. NSCLC Tissues and its Cell Lines63 cases with NSCLC and adjacent normal tissue specimens used forimmunohistochemical assay. 31 fresh lung cancer tissue and surrounding normal lungtissue specimens preserve for RT-PCR in -70℃after quick-frozen in liquid nitrogenimmediately. 3 NSCLC cell lines including human lung adenocarcinoma cell lineA549, one subseries of giant cell lung cancer with high invasiveness potential-BE1 andanother subseries of giant cell lung cancer with low invasive potential-LH7 were usedfor immunofiuorescence technology, RT-PCR and Western blot.2. Semi-quantitative RT-PCRRNAout used to extract the total RNA, synthesis of BAMBI and primer ofGAPDH. To synthesis cDNA chain by two-step RT-PCR kit, then amplification. Thegel electrophoresis imaging analysis system were used to image acquisition ofproduction by 2% agarose gel electrophores, then we calculated the integral opticaldensity value of the band.3. Immunohistochemistry ProtocolTissue by the formalin-fixed, paraffin-embedded sections prepared forimmunohistochemical staining. The primary antibody was diluted 1:200, coloration byDAB, nuclear restaining with hematoxylin, neutral gum mount the coverglass. Themicroscope with a digital CCD camera observed and captured the images.4. Cell CultureA549 cells were cultured in the presence of 10% fetal calf serum DMEM. BE1and LH7 cells were cultured in the presence of 10% fetal bovine serum in RPMI 1640medium. All the cells are cultured at 37℃, 5%CO₂ incubation conditions. 5. Western blot ProtocolCentrifugal collection of cells adding some lysate, take the supernatant. AfterSDS-PAGE electrophoresis, transfer the membrane, incubation in primary antibody(1:1000) -4℃overnight after blockage. Incubation in HRP-conjugated secondaryantibody (1: 10000) for 2h at room temperature next day, using ECL reagent indarkroom and exposure, Gel electrophoresis imaging analysis system for capturing theimage and calculating integral optical density value of the bands.6. Immunofluorescence ProtocolCells labeled 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 restaining with PI (1:1000),mounting. Application of laser scanning confocal microscope image acquisition systemobservation.ResultsThe level of BAMBI mRNA in cancer tissues was higher than that in thecorresponding adjacent tissues (0.358±0.135vs0.249±0.129), with the differencebeing statistically significant (P=0.003). BAMBI protein expressed mainly in themembrane and the cytoplasm close to the membrane, its expression in the cancer tissuewas higher than that in the adjacent tissues, the difference was significant (P＜0.01).BAMBI mRNA expression in the A549 was lower than that in BE1 and LH7. (0.574±0.035, 0.731±0.051,0.734±0.049). Respectively, the difference was significant (P＜0.001) and BAMBI mRNA expression were no significant differences statisticallysignificant in BE1 and LH7 (P=0.216). The relative amount of BAMBI protein inBE1, LH7 and A549 were 0.769±0.103, 0.963±0.061, 0.957±0.048.A549 proteinexpression was significantly lower than that in BE1 and LH7 (P＜0.001), and relativeamount of BAMBI protein were no significant differences statistically significant inBE1 and LH7 (P=0.096). The BAMBI expression in A549 cells membrane stainingshowed weakly and granulo-ununiformity while BAMBI expression in BE1 and LH7membrane stained strongly, continuous and uniform. ConclusionThe abnormally high expression of BAMBI has relationship to non-small cell lungcarcinogenesis, so the expression of BAMBI in adenocarcinoma of lung is higher thanthat in squamous carcinoma and lower than that in giant cell lung carcinoma. Theexpression of BAMBI has no relationship to invasion potentiality of giant cell lungcarcinoma.