Study On The Over-expressed Protooncogenes And Biological Characteristics Of HER2/neu Induced By Anti-BPDE

Benzo(a)pyrene (B[a]P) as a member of polycyclic aromatic hydrocarbons (PAHs) is a ubiquitous environmental contaminant, which is formed during pyrolisis and the incomplete combustion of organic materials. It is mainly found in tobacoo smoke, broiled foods, diesel exhausts, and polluted environments, and induces various pathogenic effects in vivo, especially carcinogenesis. Tumor epidemiology investigations and laboratory studies have found that B[a]P is a strong carcinogen relative to tumorigenesis, especially to human lung cancer. In 2006,B[a]P has already identified as the first kind"human carcinogens"not a member of the second kind A group"possible human carcinogens"by International Agency for Research on Cancer,IARC. BaP is an indirect carcinogen,which has not strong carcinogenesis until it need to be metabolized into activate ultimate matters in vivo. The complex process of BaP metabolism may produce more than 20 kinds of oxide and amounts of combos, such as epoxide, hydroxybenzene, benzoquinone, diol, dihydrodiol, diolepoxide and tetrol etc, among which anti-7,8-dihydrodiol-9,10-epoxide benzo(a)pyrene(anti-BPDE) is the most strongest carcinogenesis. anti-BPDE exhibits an electron affinity to predominantly reacted with proteins and nucleic aid, especially reacted with purine resulting in BPDE-DNA adducts formation, DNA mutation,oncogenes and/or repressor genes expression or function alteration. Moreover, anti-BPDE may combinate with proteins and transcription factors to influence cells metabolism and cell cycle process, inducing cells malignant transformation. Although there are some researchs on BaP carcinogenesis, its molecular mechanisms is still unclear. Therefore, the study try to first detect expressions of proto-oncogenes which are found to be over-expressed in human lung cancer via searching for references at the levels of transcription and translation in anti-BPDE transformed cells (16HBE-T). Second, we attempt to carry out RNA interference technology to inhibit the expression of oncogene HER2/neu which is verified to overexpression in 16HBE-T, and then effects of inhibition of HER2/neu expression on anti-BPDE transformed cells biologic characters is observed. This study not only provides some evidences for exploring the molecular carcinogenesis mechanisms of environmental contamination B[a]P, but also offers some theories and techniques basis for gene prevention, forepart diagnosis and therapy of tumor. These results report as following:PartⅠStudy on the expression of main protooncogenes induced by anti-BPDEObjective benzo[a]pyrene (B[a]P) is combustion-related environmental pollutants and include known carcinogens. Anti-benzo (a) pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (anti-BPDE) is the most strong carcinogen among activated metabolites of B[a]P. Though some researches have been carried out, it is not very clear about the carcinogenesis mechanism of anti-BPDE. This study is carried out to investigate expressions of several main proto-oncogenes which were reported over-expressed in human lung cancer in the anti-BPDE-transformed 16HBE cells model (16HBE-T).Methods We analyzed 4 oncogenes overexpressed in human lung cancer by searching amounts of literatures, and chose oncogenes possibly associated with environmental pollutants benzo[a]pyrene. Total RNA and proteins were extracted from anti-BPDE malignant transformed 16HBE cells (16HBE-T) and 16HBE cells treated by solvent DMSO (16HBE-N) respectively.The levels of HER2/neu, c-myc, cyclin D1, K-ras mRNA and protein expression were analyzed by RT-PCR and Western blot assays. The location of these proteins expression was detected by immunocytochemistry.Results RT-PCR and QRT-PCR analysis demonstrated that the mRNA level of these oncogenes (eg. HER2/neu,c-Myc,cyclin D1 and K-ras) expression were different between 16HBE-T and 16HBE-N cells. The proteins level of above oncogenes was detected to be overexpression by Western blot analysis, and those proteins location were investigated to be normal by immuocytochemistry technique.Conclusion We have screened and identified some main overexpression protooncogenes in malignant transformed 16HBE cells induced by anti-BPDE. These protooncogenes activation may be one of molecular mechanisms about anti-BPDE carcinogenesis. It has provided some data with the further studies for human lung cancer induced by environmental pollutants benzo[a]pyrene.PartⅡStudy on the inhibition of HER2/neu in anti-BPDE transformed cellsObjective: The previous study revealed that proto-oncogene HER2/neu was detected to be overexpressed in the malignant transformed 16HBE cells induced by anti-BPDE. For a better understand of the role of HER2/neu in the anti-BPDE transformed cell, we attempted to carry out the inhibition of HER2/neu in the 16HBE-T using RNA interference technology. First, we designed three target sequences of silencing HER2/neu and obtained three siRNAs targeting against HER2/neu by chemical synthesis assay. The aim of this part is to screen the effective target sequences of silencing HER2/neu and establish stable HER2/neu knockdown 16HBE-T cells by retroviral delivery shRNA. This is a basis of a further investigation to the effect of inhibition of HER2/neu in the anti-BPDE transformed 16HBE cells.Methods: The full sequence of HER2/neu mRNA was gained from GenBank. We designed three different effective HER2/neu target sequences for RNA interference using bioinformaticology and obtained those siRNA for HER2/neu by chemical synthesize assay, and then transfected 16HBE-T cells with siRNAs by lipofectin. The transient tansfection efficiency was estimated by a fluorescence control (FAM-siRNA). The inhibition of HER2/neu expression was investigated by reverse transcript fluorescence quantitive PCR (QRT-PCR) and western blot technique. The effective target sequence verified was designed to construct an expressing HER2/neu shRNA plasmid (pSIREN-RetroQ-neu), which transfected RetroPackTM PT67 cells to produce some viral particles carrying HER2/neu shRNA for infecting cells. The stable 16HBE-T infected colonies were obtained by puromycin selecting culture.Results: We successfully inhibited HER2/neu expression in 16HBE-T cells using RNA interference and construct a retrovirous vector expressing HER2/neu shRNA. A stable HER2/neu knockdown 16HBE-T cell line (HBE-T-neu) was established by infecting 16HBE-T cells with viral particles carrying HER2/neu shRNA which were produced by RetroPackTM PT67 cells transfected.Conclusion: We had screened special effective target sequences for HER2/neu siRNA, and quickly constructed an expressing HER2/neu shRNA retrovirous vector, and then obtained a stable HER2/neu knock down 16HBE-T cell line (HBE-T-neu). This study has provided some data for exploring the role of HER2/neu oncogene in the anti-BPDE-transformed cells.PartⅢEffects of inhibition of HER2/neu on anti-BPDE transformed cellsObjective This study was based on the system of HER2/neu down-regulation in anti-BPDE transformed cells established by RNA interference. We investigated the cell biologic phenotype and the effects on proteins expression of its relative protooncogenes in the above established HER2/neu knockdown system.Methods We applied flow cytometry, MTT assay and anchorage-independent colony growth in soft agar test respectively to detect cell cycle, cell apoptosis, cell viability and growth in soft agar following down-regulation of HER2/neu by RNAi in 16HBE-T cells. In addition, the levels of c-Myc and cyclin D1 proteins were also measured by western blot assay after the down-regulation of HER2/neu using two different effective siRNA.Results Down regulation of HER2/neu in 16HBE-T cells significantly increased the proportion of cells in G0/G1 phase (67.09%±2.12%) and decreased the proportion in S phase (17.25%±4.14%) and exhibited significantly lower viability over 7 days of culture than negative control cells(HBE-T-N) or empty control (16HBE-T). The expressions of c-Myc and cyclin D1 protein were significantly down-regulated (～60%) after inhibition of HER2/neu using two different effective siRNAs in the 16HBE-T.Conclusion These findings suggested that inhibition of HER2/neu in anti-BPDE-transformed 16HBE cells may reverse partly cell malignant phenotype. HER2/neu might play a vital role in the malignant transformed 16HBE cells by anti-BPDE.