To Study The Reiationship Of BRF2 Protein To The Tumor Invasion And Metastasis And Prognosis In Non-small Cell Lung Cancer And Esophageal Cancer

Background and significanceChinese cancer registration report shows that Chinese common tumor were lung cancer, gastric cancer, rectal cancer and esophageal cancer compared with developed countries.And lung cancer is seriously threatening the human life and health for a long time.Especially in the recent decades,air pollution and smog has led to increased risk of lung cancer with the development of industry. In addition, esophageal cancer is high incidence cancer in China, and the survival rate of the patients is lower.The main treatment of cancer are operation treatment, radiation therapy, chemical therapy, Chinese medicine therapy, immune therapy and the rapid development targeted therapy in recent years, but most patients diagnosed in advanced, the effective treatment time has been lost. At present, all various methods of treating lung cancer all are not satisfactory,resulting in the 5 year survival rate and disease free survival rate of lung cancer and esophageal cancer patients have not improved obviously in recent years.Invasion and metastasis are the primary causes of death in patients with malignant tumors.Cell proliferation (the process cancer cells spread from the primary tumor to distant organs) and their relentless growth is a most terrible side.Although in the operation technology, basic nursing, patient timely diagnosis and treatment of modern medicine have made great improvement, the death of patients for the invasion and metastasis caused by cancer cells is still the biggest enemy that the patients and doctors face to together. Although in recent years there is any substantive great progress on the tumor treatment theory of invasion and metastasis,understanding the molecular mechanism of tumor metastasis and the appropriate treatment strategies is still an important aspect of cancer researchers concern in the molecular gene level. The process of tumor cells away from the solid tumor tissue involving multiple biological steps,including the decrease of adhesion between tumor cells, tumor cells to escape from the basement membrane,extra cellular matrix degradation, tumor cells into the micro vascular and lymphatic micro-metastasis of tumor cells, formation and immune formation and metastatic tumor micro-environment escape.Interactions between tumor cells and the surrounding micro-environment is a dynamic process of continuous, multistage extremely complex. How to block the above process of tumor cell invasion and metastasis is an important issue to prevent tumor cell invasion and metastasis.Like normal tissues, tumors require sustenance in the form of nutrients and oxygen as well as an ability to evacuate metabolic wastes and carbon dioxide. The tumor-associated musculature, generated by the process of parthenogenesis, addresses these needs. During embryologists, the development of the vesiculate involves the birth of new epithelial cells and their assembly into tubes (parthenogenesis) in addition to the sprouting (parthenogenesis) of new vessels from existing ones. Following this parthenogenesis, the normal vesiculate becomes largely quiescent. In the adult, as part of physiologic processes such as wound healing and female reproductive cycling, parthenogenesis is turned on, but only transiently. In contrast, during tumor progression, an"carcinogenic switch"is almost always activated and remains on, causing normally quiescent vesiculate to continually sprout new vessels that help sustain expanding neoplastic growths.The forming process of angiogenesis is also an important process of the development of the metastasis tumor,but recent studies have found that angiogenesis promote the metastasis tumor’s growth to certain extent. At the same time, metastasis tumor growth promote the tumor angiogenesis. And this two-way adjustment process is a wide space,it is not just to stimulate the generation of some metastatic tumor.This is likely to be the reason that Invasion and metastasis occurs, the body will be difficult to control multiple metastases. Tumor angiogenesis are regulated by many factors, including promotion of angiogenesis factors and inhibition of angiogenesis factors. These regulatory factors are finally combined with vascular endothelial regulatory protein. Therefore, cutting off the signal to the tumor angiogenesis regulation pathway or tumor angiogenesis accepted ways of generating will inhibit tumor angiogenesis, and invasion and metastasis of tumor will be control in a certain extent.Thus, the therapy that anti-angiogenesis in tumor will be the new way of inhibiting the growth of tumor has become an important strategy in the field of modern tumor therapy.BRF2 is a gene encoded BRF2 protein, located on chromosome 8p12 location; BRF2 protein is a subunit located on the transcription factor TFⅢB and takes part in the production of small RNA catalyzed by the RNA pol Ⅲ. Relationship between BRF2 gene and TFⅢB determines its important role in tumorigenesis and development process. In recent years, more and more scholars pay attention to the important function of BRF2 protein in the angiogenesis process of malignant tumor. The study found that the high expression of BRF2 gene in breast cancer, gastric cancer, renal cell carcinoma, melanoma and lymphoma were detected. By comparing the hybridization proved that local amplification of chromosome 8p12 plays an important role in the occurrence and development process of the 40% lung squamous cell carcinoma. Further research pointed out that there is a correlation between the clinical results and the BRF2 over-expression, BRF2 can be frequently activated in the preceding stage of the invasion in the squamous cell lung carcinoma. Studies suggest that BRF2 protein may plays an important role in the invasion and metastasis process of the tumor, and BRF2 protein may be have an important regulatory function in the key steps of tumor angiogenesis.In order to further explore the relationship between BRF2 gene and the invasion and metastasis of non small cell lung cancer and esophageal cancer,we conducted a study in four aspects, (1) Detect the expression levels of BRF2 protein in patients with stage Ⅰ non-small cell lung cancer, investigate the relationship of BRF2 protein to the clinicopathologic factors, tumor angiogenesis and prognosis; (2) Detect the expression levels of BRF2 protein in patients with esophageal cancer, evaluate the relevance of BRF2 protein expression to the tumor progression, angiogenesis and prognosis.(3) Suppress BRF2 protein expression in non-small cell lung cancer cells by RNA interference in vitro, investigate the roles of BRF2 protein in regulating migration, invasion and angiogenic ability; (4)Suppress BRF2 protein expression in non-small cell lung cancer cells by RNA interference in vitro, investigate the roles of BRF2 protein in EMT process.Part Ⅰ The relationship of BRF2 protein to the tumor angiogenesis and prognosis in patients with early stage non-small cell lung cancerObjective: To detect the expression levels of BRF2 protein in patients with early stage non-small cell lung cancer, and investigate the relationship of BRF2 protein to the clinicopathologic factors, tumor angiogenesis and prognosis.Methods:One hundred and seven patients with pathologic early stage non-small cell lung cancer that successfully underwent curative surgical resection plus systematic lymph node dissection from January 2004 through October 2006 at the Department of Thoracic Surgery, Qilu Hospital, Shandong University, were enrolled in this study. Immunohistochemical staining and RT-PCR for BRF2 was performed to detect the BRF2 expression on protein and gene levels, and intratumoral microvessel density (MVD) was recorded by counting CD34-positive immunostained endothelial cells. All statistical analyses were performed with SPSS 13.0 statistical software. The x2 test was performed to examine the association between BRF2 protein, MVD and variable clinicopathologic factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the statistical significance of difference between the survivals of patient subgroups. Multivariate Cox regression analysis was used to identify the significantly independent prognostic factors.Results:The positive rate of BRF2 protein expression were 55.14%,36.96% and 34.38%, respectively, in non small cell lung cancer tissues, paracancerous tissues and normal lung tissues,The positive expression of BRF2 protein in cancer tissues was significantly higher than that in normal lung tissue and cancer adjacent tissue(x2=6.756, P=0.034), no statistically significant correlation was found between BRF2 protein expression and any clinicopathologic factors (P>0.05). High MVD was detected in 56.07% cases, and significantly associated with the depth of tumor invasion(P=0.013). The level of BRF2 protein expression in 12 fresh lung cancer tissue and paracancerous tissues were detected by Western blot and RT-PCR,,results showed that the expression of BRF2 protein 8 out of 12 in the carcinoma tissues were higher than that in paracancerous tissues in protein and mRNA level.The x2 test showed that BRF2 protein overexpression was significantly associated with high MVD (P＜0.01). Univariate survival analysis showed that patients with BRF2 protein overexpression and high MVD had a significantly poor disease-free survival and at 5 years after operation, respectively. Multivariate Cox regression analysis showed that high MVD and BRF2 proteinan were all the independent prognostic factor for unfavorable disease-free survival (P=0.008 and P=0.006) and overall (P=0.034 and P=0.036), respectively.Conclusions:BRF2 protein overexpression is common in early stage non-small cell lung cancer, and significantly associated with tumor angiogenesis and poor survival. These findings suggest that BRF2 protein may have clinical potential as a promising predictor to identify individuals with poor prognostic potential and a possible novel target molecular of antiangiogenic therapy for patients with non-small cell lung cancer. independent prognostic factor for unfavorable disease-free survival (P=0.008 and P=0.006) and overall (P=0.034 and P=0.036), respectively.Conclusions:BRF2 protein overexpression is common in early stage non-small cell lung cancer, and significantly associated with tumor angiogenesis and poor survival. These findings suggest that BRF2 protein may have clinical potential as a promising predictor to identify individuals with poor prognostic potential and a possible novel target molecular of antiangiogenic therapy for patients with non-small cell lung cancer.Part II The relationship of BRF2 protein to the tumor angiogenesis and prognosis in patients with esophageal cancerObjective: To detect the expression levels of BRF2 protein in patients with esophageal cancer, and investigate the relationship of BRF2 protein to the clinicopathologic factors, tumor angiogenesis and prognosis.Methods:Ninety one patients with pathologic esophageal cancer that successfully underwent subtotal esophagectomy and esophago-gastric anastomosis plus regional lymph node dissection from January 2002 through August 2003 at the Department of Thoracic Surgery, Qilu Hospital, Shandong University, were enrolled in this study. Immunohistochemical staining for BRF2 and CD34 was performed using the streptavidin-peroxidase method, and MVD was recorded by counting CD34-positive immunostained endothelial cells.All statistical analyses were performed with SPSS 13.0 statistical software. The x2 test was performed to examine the association between BRF2 protein, MVD and variable clinicopathologic factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the statistical significance of difference between the survivals of patient subgroups. Multivariate Cox regression analysis was used to identify the significantly independent prognostic factors.Results:The positive rate of BRF2 protein expression were 58.85%,33.33% and 30.63%, respectively, in esophageal cancer tissues, paracancerous tissues and normal esophageal tissues. The positive expression of BRF2 protein in cancer tissues was significantly higher than that in normal lung tissue and cancer adjacent tissue(x2=6.304, P=0.043), and the overexpression of BRF2 is significantly correlated with invasion depth (P=0.039). High MVD was detected in 39.56% cases and significantly correlated with smoking (P=0.044) and clinical stage(P=0.001). The x2 test showed that BRF2 protein overexpression was significantly associated with high MVD (P=0.007). Univariate analysis demonstrated that the high MVD (P=0.003), BRF2 protein overexpression (P=0.001), developing clinical stage (P<0.001), poor differentiation (P<0.001) and higher rate of lymph node metastasis (P＜0.001) significantly predicted decreased overall 5-year survival and a higher risk of recurrence.Furthermore, the multivariate analyses identified BRF2 protein overexpression (P=0.009), MVD (P=0.002) and the lymph node metastasis (P=0.013) as independent prognostic factors for progression-free survival. However, the BRF2 protein overexpression and the high MVD retained its significance as an independent prognosticator for distasteful overall as well as progression-free survival (P=0.005 and 0.028, respectively).Conclusions:BRF2 protein overexpression is common in esophageal cancer, and significantly associated with tumor angiogenesis and poor survival. These findings suggest that BRF2 protein may have clinical potential as a promising predictor to identify individuals with poor prognostic potential and a possible novel target molecular of antiangiogenic therapy for patients with esophageal cancer. tissue(x2=6.304, P=0.043), and the overexpression of BRF2 is significantly correlated with invasion depth (P=0.039). High MVD was detected in 39.56% cases and significantly correlated with smoking (P=0.044) and clinical stage(P=0.001). The x2 test showed that BRF2 protein overexpression was significantly associated with high MVD (P=0.007). Univariate analysis demonstrated that the high MVD (P=0.003), BRF2 protein overexpression (P=0.001), developing clinical stage (P<0.001), poor differentiation (P<0.001) and higher rate of lymph node metastasis (P＜0.001) significantly predicted decreased overall 5-year survival and a higher risk of recurrence.Furthermore, the multivariate analyses identified BRF2 protein overexpression (P=0.009), MVD (P=0.002) and the lymph node metastasis (P=0.013) as independent prognostic factors for progression-free survival. However, the BRF2 protein overexpression and the high MVD retained its significance as an independent prognosticator for distasteful overall as well as progression-free survival (P=0.005 and 0.028, respectively).Conclusions:BRF2 protein overexpression is common in esophageal cancer, and significantly associated with tumor angiogenesis and poor survival. These findings suggest that BRF2 protein may have clinical potential as a promising predictor to identify individuals with poor prognostic potential and a possible novel target molecular of antiangiogenic therapy for patients with esophageal cancer.Part III The roles of BRF2 protein in regulating migration and invasion ability of non-small cell lung cancer cells in vitroObjective:To suppress BRF2 protein expression in non-small cell lung cancer cells by RNA interference in vitro, and investigate the roles of BRF2 protein in regulating migration, invasion and angiogenic ability.Methods:The expression levels of BRF2 mRNA and protein were detected in non-small cell lung cancer cell lines A549, H292, H460 and 95-D by real time PCR and western blot, respectively. BRF2 siRNA was transfected into the cell line with the highest expression level of BRF2 gene by liposome 2000, fluorescently labeled negative control sequence was used to detect the transfection efficiency, real time PCR and western blot were used to detect the inhibition effect of siRNA on the expression of BRF2 mRNA and protein. Wound healing assay was performed to detect cell migration ability; Transwell invasion assay was performed to detect cell invasive ability; Tube formation assay was performed to detect cell angiogenic ability.Results:Different expression levels of BR.F2 mRNA and protein were detected in these 4 non-small cell lung cancer cell lines, of which A549 cells line showed the highest expression level. Forty-eight hours after BRF2 siRNA transfection, the results of real time PCR and western blot showed that the expression of BRF2 mRNA (P＜0.01) and protein (P<0.01) in A549 cells were both inhibited significantly. After BRF2 protein expression was decreased by siRNA, wound healing assay showed that the migration ability of A549 cells was significantly inhibited (P<0.01); Transwell invasion assay showed that the invasive ability of A549 cells was significantly inhibited (P<0.01).Tube formation assay showed that the angiogenic ability of A549 cells was significantly inhibited (P<0.01).Conclusions:Inhibition of BRF2 protein expression could effectively down-regulate the migration, invasive and angiogenic abilities of A549 cells in vitro, suggesting that BRF2 protein plays important regulating roles in promoting migration, invasive and angiogenic abilities of A549 cells, BRF2 protein might be a potential novel target of antiangiogenic therapy for patients with non-small cell lung cancer. expression of BRF2 mRNA and protein. Wound healing assay was performed to detect cell migration ability; Transwell invasion assay was performed to detect cell invasive ability; Tube formation assay was performed to detect cell angiogenic ability.Results:Different expression levels of BR.F2 mRNA and protein were detected in these 4 non-small cell lung cancer cell lines, of which A549 cells line showed the highest expression level. Forty-eight hours after BRF2 siRNA transfection, the results of real time PCR and western blot showed that the expression of BRF2 mRNA (P＜0.01) and protein (P<0.01) in A549 cells were both inhibited significantly. After BRF2 protein expression was decreased by siRNA, wound healing assay showed that the migration ability of A549 cells was significantly inhibited (P<0.01); Transwell invasion assay showed that the invasive ability of A549 cells was significantly inhibited (P<0.01).Tube formation assay showed that the angiogenic ability of A549 cells was significantly inhibited (P<0.01).Conclusions:Inhibition of BRF2 protein expression could effectively down-regulate the migration, invasive and angiogenic abilities of A549 cells in vitro, suggesting that BRF2 protein plays important regulating roles in promoting migration, invasive and angiogenic abilities of A549 cells, BRF2 protein might be a potential novel target of antiangiogenic therapy for patients with non-small cell lung cancer.Part IV The role of BRF2 protein in the process of EMT in non small cell lung cancerObjective:To detect the expression levels of BRF2 protein and EMT related protein in patients with non-small cell lung cancer, and investigate the relationship of BRF2 protein to the expression of EMT related protein. To suppress BRF2 protein expression in non-small cell lung cancer cells by RNA interference in vitro, and investigate the roles of BRF2 protein in the process of EMT in non small cell lung cancer.Methods:Seventy seven patients with pathologic non-small cell lung cancer that successfully underwent curative surgical resection plus systematic lymph node dissection from January 2005 through December 2006 at the Department of Thoracic Surgery, Qilu Hospital, Shandong University, were enrolled in this study. Immunohistochemical staining for BRF2 protein and EMT related protein were performed using the streptavidin-peroxidase method. All statistical analyses were performed with SPSS 13.0 statistical software. Mann-Whitney U test was performed to examine the association between BRF2 protein and EMT related protein.To suppress BRF2 protein expression in non-small cell lung cancer cells by RNA interference in vitro, fluorescently labeled negative control sequence and western blot were used to detect the transfection efficiency, real time PCR and western blot were used to detect the inhibition effect of siRNA on the expression of BRF2 mRNA and protein.Using Western blot technology and RT-PCR technology to detect the expression of BRF2 and EMT related protein in the level of mRNA and protein in non small cell lung cancer cell line A549 and SK-MES-1, and to examine the association between BRF2 protein and EMT related protein.Results:There is a correlation between the expression of BRF2 and EMT related protein (E-cadherin, N-cadherin protein:P=0.048 and 0.045), there is no statistical significance between the expression of BRF2 protein and snail protein(P=0.101).After BRF2 protein expression was decreased by siRNA in vitro, The western blot were used to detect the expression of BRF2 protein in A549 cells line and SK-MES-1 cells, and the result revealed that the expression of BRF2 protein is reduce, meanwhile, the expression of E-cadherin protein increased and the expression of E-cadherin protein and Snail protein decreased.Conclusions:There is a correlation between the expression of BRF2 and EMT process in non-small cell lung cancer; BRF2 protein overexpression promotes tumorigenesis in EMT process.