The Mechanism Study On The Cancerous Transformation Of Bronchial Epithelial Cells Induced By The Downregulation Of RBM5 Regulated By Cigarette Smoke PM2.5

Background:Recently, many researchers paid more attentions to the association between air pollution and respiratory system disease. In the past few years, levels of smog have increased throughout China resulting in the deterioration of air quality, raising worldwide concerns. PM2.5(particles less than 2.5 micrometers in diameter) can penetrate deeply into the lung, irritate and corrode the alveolar wall and bronchial epithelial cells, and consequently impair lung function, even induce lung cancer.Cigarette smoking is by far the most important risk factor in the development of lung cancer. Cigarette smokers have a 15-to 30-fold higher relative risk of developing lung cancer compared with nonsmokers, and 90% of all lung cancers are caused by smoking. Cigarette smoke contains about 5,000 chemicals, 73 of which have been evaluated as carcinogens. Nicotine and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) are two major components in cigarette smoke that significantly stimulate survival of both normal human lung epithelial and lung cancer cells. Burning cigarette smoke is rich in PM2.5, which is the culprit and the main source of indoor PM2.5 pollution. The carcinogenic effect of cigarette smoke PM2.5should be paid more attention. As an important part of environmental air PM2.5,currently the study on cancerous transformation of bronchial epithelial cells and pathogensis of lung cancer induced by cigarette smoke PM2.5 is a hotspot in the world. However, the fine mechanism of cancerous transformation of bronchial epithelial cells induced by cigarette smoke PM2.5 rarely understood, we will conduct a preliminary exploration in this respect.RNA-binding motif protein 5(RBM5), also known as LUCA-15 or H37, is a nuclear RNA binding protein widely distributed in mammalian tissues. RBM5 was initially cloned as a putative tumor suppressor gene mapping to the human chromosomal locus 3p21.3 that is frequently deleted in human cancers. Previous studies have shown that the expression of RBM5 was frequently reduced in different cancers, including ras-transformed Rat-1 embryonic fibroblastic cells, breast cancer,prostate cancer, human vestibular schwannoma, and primary lung cancer specimens.A number of lines of evidence indicate involvement of RBM5 and its splice variants in apoptosis, cell proliferation, and oncogenesis. It is also reported that RBM5 is closely related to the development of smoking related lung cancer. But its specific regulation mechanism is still not reported at present. Wnt signaling is a key regulator of multiple aspects of tissue development. It has emerged as a critical pathway in lung carcinogenesis as already demonstrated in many cancers. Studies have shown that cigarette smoke and nicotine are factors that induces Wnt/β-catenin activation. In our previous study, the experssion level of β-catenina was up-regulated after exposuring cigarette smoke extract(CSE) in A549 cells, and RBM5 is involved in the regulation of Wnt/β-catenin pathway. Therefore, we hypothesized that RBM5 might be involved in the process of cigarette intervention and play an important regulatory role.However, the effect of cigarette smoke on RBM5 expression in bronchial epithelial cells is not clear, and it should be further to determine whether RBM5 involves in cigarette smoke-induced cancerous transformation of bronchial epithelial cells. The present study focused on the role of RBM5 in the regulation of cigarette smoke PM2.5-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. These findings provide a scientific basis for the further clarify smoking related lung cancer molecular pathogenesis, meanwhile, it also provides new ideas for the prevention of air pollution induced lung cancer.Methods:1. Cigarette smoke extract(CSE) induces the cancerous transformation of bronchial epithelial cells BEAS-2B and the expression level of RBM5.We evaluated the effect of CSE on non-cancerous human bronchial epithelial BEAS-2B cells by exposure to varying concentrations of CSE(0, 25, 50 and 100μg/ml, respectively) or DMSO(CON) for 8 days, followed by a recovery period of 2weeks.(1)After exposure to CSE, the proliferative changes of five cell populations were examined by the MTT assay.(2)The morphology of each individual group of cells was examined under a phase contrast microscope.( 3) Invasion and migration were evaluated by a transwell and scratch test assays.(4)We examined the protein and m RNA levels of various oncogenes, including K-ras, C-myc and Cyclin A and Cyclin D1, in the CSE-transformed BEAS-2B cells.(5)We analyzed the ability of the CSE-transformed BEAS-2B cells to induce xenograft growth.(6)We determined the expression of RBM5 in these cells exposed to CSE at varying concentrations at the m RNA and protein levels.2.The effect of RBM5 overexpression on cell cycle, apoptosis, invasion and migration, and the Wnt/β-catenin signaling pathway of CSE-transformed BEAS-2B cells.(1)The T3 cells(CSE induced cancerous transformation cells)were infected with recombinant lentiviral vectors expressing GFP, LV-RBM5 or vector alone(LV-GV287). Using fluorescence microscopy, the GFP expression was analyzed 3days after the infection, and it was observed that the efficiency of the infection.(2)Analysis of the effect of RBM5 overexpression on cell proliferation and colony formation of T3 cells(CSE induced cancerous transformation cells) by MTT and Colony Formation assay.(3)The cell cycle of each group was examined by flow cytometry after the expression of RBM5.(4)The apoptosis of each group was examined by flow cytometry after the expression of RBM5.(5)Invasion and migration of each group were evaluated by a transwell and scratch test assays.(6)The expression and distribution of β-catenin in RBM5 after overexpression of T3 cells was detected by immunofluorescent labeling.(7)Luciferase reporter gene was detected to express the activity of transcription factor TCF in RBM5 after overexpression of T3 cells.( 8)The expression of cell cycle, apoptosis, invasion and metastasis related genes in each group cells were detected by real-time-PCR and Western blot.(9)The protein expression of Wnt/β-catenin signal related components were detected by Western blot after RBM5 overexpression.3.Analysis of the effects of RBM5 overexpression on the xenograft growth of CSE-transformed BEAS-2B cells(T3 cells).( 1) Establishment the RBM5 overexpression of T3 cells in nude mice subcutaneously transplanted tumor model. Tumor volume of these tumors was observed.( 2) The protein expression of cycle, apoptosis, proliferation, invasion and migration-regulated genes and Wnt/β-catenin signaling pathway related components of tumor tissue were detected by western blot.( 3) The protein expression of cycle, apoptosis, proliferation, invasion and migration-regulated genes and Wnt/β-catenin signaling pathway related components of tumor tissue were detected by immunohistochemistry.Results:1. We evaluated the effect of CSE on non-cancerous human bronchial epithelial BEAS-2B cells by exposure to varying concentrations of CSE(0, 25, 50 and 100μg/ml, respectively) or DMSO(CON) for 8 days, followed by a recovery period of 2weeks. The results of the MTT assay showed that the cell population acquired phenotypic changes, including enhanced cell proliferation, shorter doubling times,enhanced invasion and migration ability. The cells treated with 100 μg/ml CSE showed aberrant and condensed nuclei in addition to abnormal nuclear cytoplasmic ratios, compared to the control cells. Western blot and q PCR analysis showed that the expression of oncogenes, including K-ras, c-myc and cyclin A and cyclin D1, was significantly enhanced in the CSE-transformed BEAS-2B cells treated with varying concentrations of CSE. All mice(n=5) implanted with the transformed BEAS-2B cells exposed to 100 μg/ml CSE developed tumors with a latency period of ~2 weeks,indicating a high efficiency of murine tumorigenicity. We designated this CSE-induced cancerous transformation of BEAS-2B cells as ‘T3 cells?.2. We observed that both at the m RNA and protein levels, the RBM5 expression was reduced with an increasing CSE concentration, compared to the control group.The fold-change in the gene expression was significantly reduced in the T3 cells(CSE induced cancerous transformation cells).3. RBM5 overexpression inhibits the proliferation, colony formation, invasion and migration of T3 cells. Expression analysis of the genes implicated in cell invasion and migration revealed that RBM5 overexpression decreased the relative levels of HIF-1α, VEGF, MMP-2 and MMP-9. RBM5 overexpression induces cell cycle arrest of T3 cells at the G1/S phase. Further analysis of the expression of cell cycle regulators indicated that the T3-LV-RBM5 cells had significantly decreased relative levels of CDK4, CDK6, Cyclin D1 and Cyclin A, but increased levels of p53 and p21.RBM5 overexpression induces apoptosis of T3 cells, and significantly increased the levels of cleaved caspases-3 and-9, along with Bax expression. In contrast, it inhibited the expression of the anti-apoptotic protein Bcl-2. And overexpressing of RBM5 can inhibit the activation of Wnt/β-catenin signaling pathway in T3 cells.4. RBM5 overexpression inhibits the xenograft growth of CSE-transformed BEAS-2B cells. And RBM5 overexpression blocks the cell cycle, promots apoptosis,inhibits the expression of invasion related proteins, and inhibiting the activation ofWnt/β-catenin signaling pathway in tumor tissue. These datas are further reveal that RBM5 plays an important regulated role during CSE induced transformation of bronchial epithelial cells into the cancerous phenotype.Conclusions:We demonstrated that BEAS-2B cells exposed to 100 μg/ml CSE acquired cancerous phenotype and formed tumors in nude mice. RBM5 expression was inhibited in CSE-transformed BEAS-2B cells and that subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest,triggered apoptosis, inhibited their invasion and migration, including xenograft growth, and inhibit the activation of Wnt/β-catenin signaling pathway.Therefore, we considered that RBM5 may be a promising candidate for intervention of smoking-related lung cancer, and our findings may provide new insights into the understanding of the precise mechanism underlying the antitumor activity of RBM5. Meanwhile, it also provides new ideas for the prevention of air pollution induced lung cancer.