Role And Mechanism Of RB94 Gene In Nonsmall Cell Lung Cancer

Lung cancer(LC) remains the most common type of cancer and is the leading cause of cancer-related death worldwide,moreover the morbidity is rising significantly. The overall survival rate of lung cancer patients remains poor with a 5-year survival rate of nearly 15%.The main types of LC are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). Among all LC cases,NSCLC accounts for approximately 85%.NSCLC mainly comprises two types:squamous cell carcinoma (SCC) and adenocarcinoma(ADC).Despite the fact that great advances have been made in the therapeutic treatment methods, including surgical resection, chemotherapy,radiotherapy and biological immunotherapy for NSCLC,the prognosis is still suboptimal. Because many NSCLC patients are diagnosed at a late stage of the disease when the tumors are not resectable. Thus,it is ugently needed to look for new method of effective treatment for NSCLC.The rapid development of molecular biology and molecular genetics in recent years yields deeper understanding for molecular mechanisms of lung cancer. As we know, tumorigenesisis essentially caused by genetic abnormalities,so do lung cancers.Many carcinogenic factors can induce cell transformation and carcinogenesis via activation of oncogenesand/or inactivation of tumor suppressor genes.Thus,oncologists pay more and more attention to gene therapy.However,it becomes a problem that how to select effective gene therapy targets.The retinoblastoma (RB94) tumor suppressor gene, which lacks the NH2-terminal 112 amino acid residues of the full-length RB protein (RB110), has been identified to have marked tumor suppressor efficacy compared with wild-type RB110. The RB94 protein has been reported to remain in a hypo-phosphorylated state in transfected tumor cells and demonstrate a significantly longer half-life than the RB110 protein Preclinical studies also demonstrate that adenovirus-mediated (Ad)-RB94 gene transfer significantly suppresses the growth of human head and neck cancer, bladder carcinoma, pancreatic carcinoma and esophageal cancer in vitro and in vivo Moreover, the combination of RB94 and radiation therapy (XRT) results in synergistic tumor growth suppression of head and neck squamous cell carcinoma and lung cancer. Therefore, RB94 appears to be a promising candidate for gene therapy in NSCLC.In our previous studies,the eukaryofic expression plasmid pIRKS-RB94 was correctly constructed and transfected into the A549 cells and inoculated tumor in nude mice.It was suggested that RB94 gene transfer significantly suppressed the growth of lung adenocarcinoma in vitro and in vivo. Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to SIP.Two distinct SphK isoforms have been identified, SphKl and SphK2. SphKl can promote the growth of tumor cells and inhibit cell apoptosis. In contrast to t SphKl, SphK2 can inhibit the growth of tumor cells and promote cell apoptosis.After transfection, RB94 could efficiently inhibit expression of SphKl, and enhance expression of SphK2 in the A549 cell line. It is assumed that RB94 has marked inhibitory effect on A549 cells by the role of sphingosine kinase (Sphk),which makes the sphingomyelin metabolic pathways as the target of tumor therapy via intervention Sphkl/S1P pathway.Based on our previous studies,we constructed the recombinant adenovirus vector Ad-RB94 carrying green luorescence protein(GFP) by gateway technology.Next, Adenovirus-mediated (Ad)-RB94 gene infected A549 cells and inoculated tumor in nude mice in vitro and in vivo. Role and mechanism of RB94 gene in non-small cell lung cancer was investigated in the cell and animal level.The research was divided into three parts. Part I:Construction of recombinant adenovirus vector for RB94 gene using gateway clone technology;Part II:Effect of RB94 on cell cycle and expression of SphKs in human lung adenocarcinoma A549 cells;Part III:Inhibitory role of retinoblastoma 94 and effect on the expression of SphKs in a mouse xenograft model.Part I Construction of recombinant adenovirus vector for Retinoblastoma 94 (RB94) geneUsing Gateway technologyObjective:To obtain the full length of RB94 gene and construct human RB94 adenoviral vectors carrying green luorescence protein (GFP),and to establish foundation for a further study on RB94 fuction in the cell and animal level.Methods:All coding areas of RB94 gene were amplified by PCR technology.The attB-flanked PCR primers were designed and used to amplify RB94 gene by PCR. An entry clone was performed by a BP recombination reaction with attB-PCR products and vector carrying green fluorescent protein.Then the entry clone and the target vector Ad/CMV/V5-DEST with attRl and attR2 sites was recombined together to create the expression clone (Ad-RB94) by an efficient LR recombination reaction.After the expression clone was confirmed by PCR and sequencing. Ad-RB94 was digested with PacI and transferred into 293 cells to be packaged into adenovirus stock.Ad-RB94 was amplified by infection of 293 cells and the titer was measured. Results:The human recombinant RB94 was successfully cloned and the recombinant adenovirus vector Ad-RB94 was found to be successfully constructed via restriction enzyme digestion and sequencing methods.Then the adenovirus vector Ad-RB94 was transfected into 293 cells.Under fluorescence microscope, the transfected cells with green fluorescence protein could be observed. The titer of Ad-RB94 was up to 2×109 pfu/ml.Conclusions:Ad-RB94 was successfully constructed with gateway technology,which lays an experimental foundation for further research in vitro and in vivo.Part Ⅱ Effect of retinoblastoma 94 gene on human lung adenocarcinoma A549 cellsObjective:To investigate the effect on cell cycle and expression of SphKs of Retinoblastoma 94 in human lung adenocarcinoma A549 cells. Methods: Recombinant adenovirus vector Ad-RB94 infected lung adenocarcinoma A549 cells. And GFP fluorescence was used for detection infection efficiency. Level of RB94 mRNA and protein expression were determined using real time PCR and Western Blot method. Cell cycle distribution was measured by flow cytometry. The effect on the expression of SphKs of Ad-Rb94 was measured by real time PCR and cell immunofluorescence.Results:Compared with the blank control group and empty vector group, the number of cells of G2/M phases increased,whereas the cell number of G0/G1 phases and S phases decreased (P<0.05); Moreover,RB94 gene can inhibit expression of SphKland enhance expression of SphK2 both by real time PCR and by cell immunofluorescence.Conclusion:Ad-RB94 may induce G2/M cell cycle arrest in A549 cells.Moreover, RB94 gene down-regulated expression of SphK1 and up-regulated expression of SphK2.Part Ⅲ The role of retinoblastoma 94 gene in a mouse xenograft model of human lung adenocarcinomaObjective:To investigate the inhibitory effect of RB94 on growth of A549 xenograft tumor in nude mice.Methods:The nude mice model of human lung adenocarcinoma was established by subcutaneous injection with A549 cell suspension. The expression of RB94 was determined by real-time RT PCR. Growth inhibition of the xenograft tumor induced by RB94 was observed in vivo. Cell apoptosis was detected by Hoechst staining. The effect on the expression of SphKs of Ad-RB94 was measured by real time PCR and immunofluorescence.Results：Compared with the other two group, the volume and weight of xenograft tumor in Ad-RB94 injection group were significantly decreased and the inhibitory rate was 60.5%. Treatment with Ad-RB94 induced an apoptosis rate of 26%.Moreover,RB94 gene down-regulated expression of SphKl and up-regulated SphK2 expression both by real time PCR and by immunofluorescence.Conclusions:RB94 can inhibit the growth of A549 xenograft tumor in nude mice and induce apoptosis in vivo. Furthermore, RB94 gene inhibited expression of SphKl and enhanced expression of SphK2 in vivo.