The Study Of Impact Of CCNB1 On Cell Cycle And Proliferation In Non-small Cell Lung Cancer Cells And Potential Mechanism

Part ? The impact of CCNB1 on non-small cell lung cancer(NSCLC)cell cell cycle and proliferation and its association with clinicopathological parameters and patient prognosisObjective: To evaluate the influence of cell cyclin B1(CCNB1)on proliferation,cell cycle,apoptosis,migration and invasion in NSCLC cells.The association of CCNB1 with clinicopathological parameters was assessed with materials from patients with NSCLC and subsequently validated in microarray datasets from public database,simultaneously the survival analysis was conducted.Methods: QPCR was employed to test the expression level of CCNB1 in the manner of m RNA both in NSCLC cell lines and lung adenocarcinoma specimen.The vectors used for sh RNA interfering and overexpression of CCNB1 were constructed followed by investigation of the impact of CCNB1 on cell cycle,apoptosis,proliferation,migration and invasion.Nude mice were utilized to evaluate the ability of tumorigenesis of CCNB1 both in sh RNA and overexpression group.The microarrary datasets form TCGA and GEO were used to validate the association of CCNB1 with clinicopathological parameters and probe its relation with survival of patients with NSCLC.Results: The results of qPCR showed that the expression of CCNB1 was universally present in various NSCLC cell lines.In lung cancer specimen,the level of m RNA of CCNB1 was significantly higher than that in adjacent normal lung tissues(P <0.01).The proteins related with cell cycle and apoptosis were stable both in sh RNA and overexpressed group in western blot study.In additition,the level of expression of CCNB1 was rarely correlated with apoptosis,migration and invasion for H1299 and A549.In respect to cell cycle,the cell cycle of both H1299 and A549 was arrested in G2/M phase in sh RNA interfering group;moreover,the arrest of cell cycle in G2/M was observed in the overexpression group.As to the proliferation,the ablility of cloing formation of A549 was inhibited in sh RNA group and nude mice xenograft was suppressed in terms of volume and weight.Similar results were observed in CCNB1 overexpression group with the slightly minor impact.After both univariate and multivariate analysis,the level of CCNB1 m RNA was associated with gender,somking,T stage and N stage among lung adenocarcinoma patients and this association was validated by TCGA dataset.The survival analysis with TCGA and GEO microarray datasets demonstrated that higher level of CCNB1 expression predicted poorer survival in both overall survival and disease-free survival with significant difference.Conclusion: CCNB1 was able to affect the proliferation and cell cycle of NSCLC cells.In contrast,CCNB1 showed no impact on apoptosis,migration and invasion in NSCLC cell lines.CCNB1 was associated with clinicopathological parameters of patients with lung adenocarcinoma and high level of CCNB1 predicted dismal survival rate.Part ? Degradation of CCNB1 mediated by APC11 through UBA52 ubiquitilation promotes NSCLC cell cell cycle progression and proliferationObjective: To investigate the mechanism of CCNB1 by which the cell cycle of NSCLC cells was affected and to provide potential explanation for the relationship between CCNB1 expression and survival rate.Methods: The sample derived from coimmunoprecipitation(COIP)assay was analyzed through proteomics study to determine the proteins which interact with CCNB1.A combination of proteomics result and literature reading resulted in the decision making that UBA52 and APC11 were the objects to be investigated.Inhibition and overexpression of UBA52 were accomplished through corresponding vectors.The interaction between UBA52 and CCNB1 was assessed in terms of protein level,cell cycle and proliferation in NSCLC cell lines.Then inhibited and overexpressed APC11 cells were used to validate this interaction.Results: Proteomics study showed CCNB1 was potentially interacted with UBA52.After overexpression of UBA52,the protein level of CCNB1 decreased,cell cycle progressed and the ability of cloning formation was enhanced in NSCLC cell lines;however,after inhibiting expression of UBA52,the protein level of CCNB1 was higher,the cell cycle was arrested in G2/M phase and fewer cloning formation was observed.When overexpressing UBA52 in previously CCNB1 overexpressed NSCLC cells,the protein level of CCNB1 relatively decreased in comparison with that without UBA52 overexpression,cell cycle progressed and more cloning formation was observed,and this phenomenon was reversed by inhibiting APC11 via si RNA resulting cell cycle arresting in G2/M phase.Subsequtent overexpression of APC11 resulted in lower level of protein CCNB1 and significantly promoted cell cycle progression and cloing formation.Conclusion: Degradation of CCNB1 mediated by APC11 through UBA52 ubiquitilation promoted cell cycle progression and proliferation.