CCNB2Can Promote Cell Proliferation, Migration And Invation In Human Lung Cancer

BackgroundPrimary bronchial lung cancer is one of clinical common malignant tumors which derives from the primary bronchial mucosa and alveolar. Age standardized mortality of lung cancer increased at a speed of1-5%per year and the lung cancer mortality of female is rapidly increasing. This trend is especially pronounced in developing countries whereas in the developed country(except France and Japan) such as Europe this trend is leveled off, even in the fall. With the continuent and rapid rising of Lung cancer incidence and mortality in China, the incidence of lung cancer growing at a speed of26.9%a year at present.If we donot timely take effective measures, by2025,the number of lung cancer patients in our country will reach1million, becoming the world’s first lung cancer country. Research suggested that some factors such an smoking, environmental pollution and radon exposure can be the leading cause of lung cancer. According to the cytological and histological knowledge, this disease can be divided into squamous cell carcinoma, adenocarcinoma and large cell carcinoma, small cell carcinoma, gland scale cancer, as well as a rare type of cancer, adenoid cystic carcinoma and mucoepidermoid carcinoma, etc. Because most of the patients with lung cancer onset hiddenly,patients have been found more than in the late and the mortality is high. The formation of lung cancer involves multiple genetic and multiple signaling pathways changes, the activation of oncogene and the inactivation of tumor suppressor gene and imbalance of interaction between them.They are all the molecular basis of lung cancer development and the abnormal adjusting of cell cycle related genes expression may play an important role in the procession of cell proliferation in lung cancer. Due to the easy early metastasis by blood of lung adenocarcinoma, the distant metastases is often the cause of the failure of treatment. So the exploration of important genes associated with lung cancer and its mediated signaling pathways will help reveal the pathogenesis of lung cancer, find the molecular markers for early diagnosis of lung cancer, provide the basis of molecular biology for its treatment.CCNB2(cell cycle protein B2), is a member of the family of cell cycle protein. Cell cycle protein B group includes B1and B2which plays an important role in cell cycle regulation, mainly by combing with Cdc2to promote cell enter into M phase from the G2. Cyclin B1and Cyclin B2differs in the localization of cells. Cyclin B1locates in particles in the cytoplasm and Cyclin B2locates in the golgi apparatus before the split phase,while in a divided period,Cyclin B1enter into the nucleus and Cyclin B2is dispersed among cells. Now it is suggested that CCNB2involves in tumor progression, and thus the study of CCNB2may reveal some of the molecular mechanism of tumour. Some literatures have been written that Cyclin B2in the human large intestinal cancer has a higher expression than normal adenocarcinoma tissue; Pituitary adenoma in mice and human have high expression of Cyclin B2which is regurated through HMGA; In human lung adenocarcinoma tissue samples, there is also higher expression of CCNB2than that of normal tissues of lung cancer; And there are also literatures reporting the using of CCNB2expression level in serum as a basis for diagnosis of lung adenocarcinoma.The study of the role of CCNB2in lung cancer and its mechanism is mainly carried out through investigating the effect of CCNB2on lung cancer cell proliferation, migration and invasion.TGF beta signaling pathways can regulate cell proliferation, migration and invasion in mang tumors.It is considered that the mian function of this pathway is the inhibition of proliferation, promotion of migration and invasion in the early period of the tumor, while in the late stage of tumor, it promotes the proliferation, migration and invasion. Existing researches show that TGF beta RII can combine Cdc2through Cyclin B2and to regulate the cell cycle.PI3K/Akt signaling pathway in the tumor can regulate cell migration and invasion, but the relationship between this pathways and CCNB2, the regulating functions of migration and invasion in lung cancer are not clear. In our this subject, we studied the effects of CCNB2on the proliferation, migration and invasion in lung cancer and have a preliminary study of its molecular mechanism.ObjectiveTo identify CCNB2expression features in lung adenocarcinoma cancer cells and in lung adenocarcinoma tissues respectively and then make sure the role of CCNB2in regulation of lung cancer cell proliferation, migration and invasion and then to vertify the molecular mechanisma of all above of these roles.Contents and methods1. To identify CCNB2express feature in lung adenocarcinoma tissues and lung adenocarcinoma cancer cells respectively The mRNA levels and protein levels of CCNB2expression were respectively detected by Real-time Q-PCR and Western blot in lung cancer cell lines compared to HBE;The CCNB2expression levels in lung adenocarcinoma cancer were deteced by immunohistochemical compared to tissues adjacent to carcinoma.2. To make sure the effect of CCNB2on lung cancer cell proliferation and identify the molecular mechanism in lung cancer in vitro1) Use of packaging lentivirus constracted by company to transfect A549and Spcal, establishing cell lines with stable expression and stable interference of CCNB2; Fluorescence quantitative PCR and Western blot were used to identify the overexpress efficiency and interference efficiency.2) Capacities of proliferation in lung cancer cell lines were detecd by MTT and Tablet Cloning text after overexpressing and reducing expression of CCNB2;3) Percentage of cells in S phase of cell cycle in A549and Spcal after reducing expression of CCNB2was detected by EDU and cell cycle text;4) The change of the capacity of tumor-forming was detected by the text of formation of tumor in Nude mice in vitro after CCNB2was stability interferenced, and then getted out of the tumor, implementted tissue section and immunohistochemical detection, then observed the changes of expression quantity of cell cycle related factor;5) The change of some proteins related cell cycle in A549and Spcal after overexpressing and reducing expression of CCNB2was detected by Western blot and the related molecular mechinasm was also studyed primary;6) After instataneous interference of TGF beta in cell of A549-C1in which the expression of CCNB2was stably interferanced,we implemented a serious of text to indentify the change of proliferation abilities and detected the change of protein related cell cycle by Western blot.3. To find the effect of CCNB2on lung cancer cell migration and invasion and identify the related molecular mechanism in lung cancer in vitro1) Capacities of migration and invasion in lung cancer cell lines were detecd byTranswell and Boyden text after overexpressing and reducing expression of CCNB2;2) The change of the capacity of cell migration and invasion was detected by Scratch Text;3) The change of some proteins related EMT in A549and Spcal after overexpressing and reducing expression of CCNB2was detected by Western blot and we had a primary knowledge about the related signal pathways.Resultsl.The levels of CCNB2expression were higher in A549and Spcal cells lines compared to HBE;The CCNB2expression levels in lung adenocarcinoma cancer were higher compared to tissues adjacent to carcinoma by immunohistochemical detection.1) CCNB2expression in two lang cancer cell lines and HBE were measured by real-time quantitative PCR and Western blot,and the results have obvious differenced between the two lang cancer cell lines and the normal lung epithelial cell line HBE;2)Immunohistochemistry results showed CCNB2was mainly expressed in the cytoplasm.Expression of CCNB2in lung adenocarcinoma cancer tissues was increased compared to tissues adjacent to carcinoma.Higher expression of CCNB2was not related with tumor vascular invasion,lymph node metastasis and clinical stage.2.CCNB2promote cell proliferation in lung cancer and its molecular mechanisml)The overexpress efficiency and interference efficiency were detected by Fluorescence quantitative PCR and Western blot seperately, and the results have obvious differenced;2)After overexpressing and reducing expression of CCNB2,the abilities of cell proliferation were inhanced and inhibited respectively by using MTT and Tablet Cloning text;3)EDU proliferation assay and cell cycle text showed that Percentage of cells in S phase of cell cycle in A549and Spcalwere decreased after reducing expression ofCCNB2;4)The change of the capacity of tumor-forming was inhabitted in vitro after CCNB2was stably interferenced, the expression quantity of cell cycle related factors such as P21,P-P53increased compared to the control group;5)Western blot showed that some proteins related cell cycle such an P21,P-P53and P-Smad3, TGFβ were downregulated and upregulated in A549and Spcal after overexpressing and reducing expression of CCNB2seperately;6) After instataneous interferance of TGF beta in cell of A549-C1in which the expression of CCNB2was stably interferanced, the cell had a stronger proliferation abilities and some proteins related cell cycle such an P21,P-P53were downregulated..3. CCNB2promote cell migration and invation in lung cancer and its molecular mechanism1) Transwell and Boyden text showed that capacities of migration and invasion in lung cancer cell lines were inhanced and inhibited respectively after overexpressing and reducing expression of CCNB2;In all, CCNB2can promote cell migration and invation in lung cancer;2)After decreasing expression of CCNB2, Scratch Text suggested that cells had lower abilities of migration;3) Western blot showed that some proteins related EMT such an E-CA,ZO-1were downregulated and upregulated whereas N-CA,Snail were upregulated and downregulated in A549and Spcal after overexpressing and reducing expression of CCNB2seperately. The expression of PI3K,AKT were downgraded after interference CCNB2.CCNB2may regulate cell migration and invasion of lung cancer cells through the PI3K/AKT signal pathway.ConclusionThe expression of CCNB2in lung adenocarcinoma cancer cells lines A549, Spcal is higher than normal pulmonary bronchial epithelial cells HBE; Compared with the tissues adjacent to carcinoma, lung adenocarcinoma tissues have higher CCNB2expression; After stable overexpression and stable interference of CCNB2, the cell lines have enhancement and weaken ability of cell proliferation respectively so it suggests that CCNB2can promote lung cancer cell proliferation,this may be implemented by TGF beta signaling pathways; CCNB2can also promote lung cancer cell migration and invasion, the control may be achieved by PI3K/Akt signal pathway; The above results suggest, CCNB2can promote lung cancer cell proliferation, migration and invasion, closely related with the occurrence and development of lung cancer.