Expression And Significances Of BMP-2 And CD105 In Human Non-small Cell Carcinoma

Cancer is one of the diseases that serious threat to human health, so far, had not yet been found satisfactory management method. thus seeking new antitumor method is particularly important. Infiltration and metastasis are not only the major characteristic of the malignant tumor, but also the key to its hard curing and the resulting death of patients. Tumor blood vessel is the basis for growth, invasion and metastasis of malignant tumors. Tumor angiogenesis and tumor cell not only reflect levels of nutrition and oxygen-related, but also reflects the invasion and metastasis of tumor, and are an important factor of tumor size, transfer, recurrence and prognosis. Including most of the growth and metastasis of malignant tumor of lung cancer have a vascular-dependent feature.MVD within the tumor angiogenesis as a judge of indicator has been widely considered to be an important factor in predicting prognosis of tumor. CD 105 is expressed mainly in proliferating endothelial cell and signs angiogenesis on specific. CD 105 can be regulating the activity of the transforming growth factor-β(TGF-β) to promote endothelial cell proliferation, differentiation and migration, and participates in angiogenesis.Bone morphogenetic proteins (BMPs) are cytokines belonging to TGF-βsuper family, and are a set of widely involved in the regulation of a variety of cell proliferation, differentiation and apoptosis of the biological process of functional proteins, can induce migration and differentiation of progenitor, regulation of cell growth, proliferation, migration, and embryonic stem cell self-renewal. At present, most researchers agree that BMP-2 in the growth, invasion and migration of malignant tumors has a very important role.Presently there are only a few studies on the expression of BMP-2 in lung carcinoma at abroad and there are no related reports at home. The relation between BMP-2 and lung cancer is unclear. In order to discuss the correlation of genes of BMP-2 and MVD (marked by CD105) with growth and trans-infiltration of non-small cell lung cancer (NSCLC), find the targets of early detecting lung carcinoma growth and metabasis, and explore the effective way which could prevent its growth and metabasis, we detected BMP-2 and MVD (marked by CD105) expression in lung cancer by immunohistochemical methods and Western Blot. The expression of BMP-2 mRNA in NSCLC was detected with RT-PCR. We analyzed relationships among BMP-2, MVD and lung carcinoma, discussed the interaction between BMP-2 and MVD (marked by CD 105) in NSCLCMethods:1. The immunohistochemical staining was used to test the expression of BMP-2 protein and CD 105 in 50 cases of NSCLC,10 cases of normal lung tissues, Selected cases without any chemotherapy before surgery and radiotherapy2. Western Blot was performed to test the BMP-2 protein level in the case group and the control groups, observe the expression of BMP-2 Protein between 20 cases of NSCLC and 10 cases of normal lung tissues, and analyze the relation for the expression of BMP-2 Protein among lung cancer clinical pathology.3. RNA extraction, cDNA synthesis and PCRTotal RNA from fresh tissues was isolated with guanidinium isothiocyanate. The integrity of total RNA was identified at 90V on 1% agarosegels containing EB (0.5ug/ml).The UV Spectrophotometer was used for the quantization of the total RNA. Total RNA was reversed transcribed using reverse transcriptase with oligoDT at 43℃for 90min to synthesize cDNA. The PCR was carried out in a duplex mixture such as cDNA, TaqDNA polymerase, primers and so on, denaturation for 5 mins, 94℃50s.62℃30s.72℃30s,30 circulations. After amplification, each sample was analyzed by 1.5% agarose gel electrophoresis for 40mins. At the same time, we used GAPDH as an internal control.4. Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS), version 13.0 for windows. Chi- square test is used to compare the expression of BMP-2 protein and results of Western Blot and RT-PCR. Between groups T-test is used to test the expression of CD105, and assess the relation of BMP-2 and MVD (marked by CD 105). P-values of<0.05 were considered to be statistically significant.Result: 1. Expression of BMP-2 protein in tissues:In lung cancer and normal tissue the Positive rate of BMP-2 protein was 68% in samples of NSCLC tissues and 0% in normal lung tissues (P<0.05). In the clinical and pathological features groups of non-small cell lung cancer the positive rate of BMP-2 in groups of I and II clinical stage cases was 53.6%, lower than the expression inⅢandⅣclinical stage lung cancer(86.4%)(P<0.05). The positive rate of BMP-2 in groups of poorly differentiated lung cancer was 85%, higher than the expression in well-differentiated and moderately differentiated cases (56.7%) (p<0.05). The positive rate of BMP-2 protein in the samples of lymph node metastasis was 78.1%, higher than the expression in the samples without lymph node metastasis 50% (P<0.05). But there were no statistical significance between male and female, younger and older, squamous cell carcinoma and adenocarcinoma.2. Expression of MVD(marked by CD105) in NSCLC:MVD value of lung cancer was 41.32±4.52,in normal lung tissue was 30.12±3.21, the difference between their was significant (P<0.05). In NSCLC MVD valueⅠandⅡclinical stage cases was35.63±4.31 lower than the expression in PoorⅢandⅣclinical stage lung cancer(44.80±4.61) (P<0.05). MVD value the samples with lymph node metastasis of NSCLC was 47.31±4.94 higher than the expression in the samples without lymph node metastasis 34.61±4.19 (P<0.05). MVD value of well-differentiated and moderately- differentiated cases was 38.63±4.85, lower than the expression in poorly-differentiated lung cancer (41.27±4.23)(p>0.05). But there were no statistical significance between male and female, younger and older, squamous cell carcinoma and adenocarcinoma, well-differentiated and moderately-differentiated.3. The relationship between BMP-2 and CD 105 in NSCLC:MVD (marked by CD 105) value in the BMP-2- Positive group (42.46±3.15) was higher than BMP-2 negative group (33.28±3.21). There was significant difference between the two groups (P<0.05).4. Western Blot was performed to test the BMP-2 protein level in the case group and the control groups, observe the expression of BMP-2 protein between NSCLC and normal lung tissues, BMP-2 expression in non-small cell lung cancer is high, which in the adjacent normal tissue expression level is very low. Expression BMP-2 protein in normal tissue(0.199±0.027) is lower than that in lung adenocarcinoma (2.278±0.041) and squamous cell carcinoma of lung(1.869±0.032) (p<0.01). There are no significant differences when lung adenocarcinoma and squamous cell carcinoma compared with each other (p>0.05).5. BMP-2 mRNA expression in NSCLC:The result of RT-PCR showed that BMP-2 mRNA expression was detected in 17 of 20 NSCLC,72of 10 normal lung tissues. The positive expression rates of BMP-2 mRNA in NSCLC and normal lung tissues were 85% and 20% respectively. Between NSCLC and normal lung tissues the expression of BMP-2 mRNA was signifficant difference (p<0.05).Conclusion:1. The levels of the expressions of BMP-2 mRNA and protein in nomal lung tissues were all low that were high in NSCLC. The level of the expressions of BMP-2 protein was related to the lymph node metastasis, clinical stage and differentiation degree.The results of Western Blot and RT-PCR indicated that the high expression of BMP-2 protein and mRNA might play an important role in the development of NSCLC. The results indicated that the amount of expression of BMP-2 and pathological type have no obvious relationship in lung carcinoma.BMP-2 may interactive in the progress of the invasion, metastasis and angiogenesis and other of NSCLC.2. MVD (marked by CD105) value in NSCLC correlated with the lymph node metastasis and clinical stage. MVD may be involved in the invasion, metastasis and angiogenesis processes of lung cancer.3. The expression of BMP-2 protein and CD 105 in NSCLC showed a positive correlation with each other. Suggesting that they has a certain synergy in the progress of the angiogenesis development, and invasion of NSCLC.