| Tobacco related death has become the first cause of death worldwideand it is estimated that approximately1millions patients each year diedfrom tobacco related diseases in China,the most common diseases fromwhich are COPD and lung cancer.Recently,the effects of long-termexposure to PM2.5(particulate matter) on human health have drawn muchattention from clinicians and researches.Cigarette smoke is one of themain sources of indoor PM2.5. Cigarette smoke (including the invisiblepart) comprises tiny particles and is rich in PM2.5, which can go directlyinto the airways and is a major respiratory system hazard.PM2.5once isinhaled into the respiratory tract, can be deposited in the diameter of2-4m small airway, airway defense barrier is damaged, leading to lungfunction disorders, which cause increased incidence of respiratory systemdiseases. At the same time, cigarette smoke also includes nearly sixthousand kinds of chemical substances, most of which are harmful to thebody, especially benzopyrene.It is proved that benzopyrene is a class oforganic compounds with significant carcinogenic effect. Therefore, toexplore the cigarette smoke exposure to PM2.5induced lung injurymechanism can provide new targets and the scientific basis for theprevention and treatment of pulmonary diseases.The RBM family is a newly discovered by HUGO genenomenclature committee, officially named RRB/RBM/RNP proteinsubsets, such as protein is a RNA binding protein, may be associated withRNA splicing, translation and stability. Current studies have showed thatRBM5is a new lung cancer candidate tumor suppressor gene and its mostimportant characteristic is involvement in the regulation of cell apoptosisprocess, and is high related with the development of tobacco related lungcancer.Wnt signaling pathway exists widely in invertebrates and vertebrates, are a class of highly conserved in the evolution of species inthe process of signal pathway. Wnt signaling plays a crucial role in earlydevelopment of animal embryo, organ formation, tissue regeneration andother physiological processes.Current studies show that Wnt/β-cateninsignaling pathway is closely related with the occurrence, development oftobacco related diseases, especially the occurrence and development oflung diseases. It is found that drug resistance was reversed in human lungadenocarcinoma cell line A549/DDP,the numbers of cell apoptosisincreased and β-catenin gene expression levels reduced aftertransfecting the exogenous RBM5gene. We hypothesized that RBM5, asthe upstream β-catenin gene, Wnt/β-catenin signaling pathway in tobaccorelated lung injury.This experiment conducted in human alveolar epithelialcells(A549),targeted RBM5-Wnt/β-catenin pathway, to explore themolecular regulation mechanism of RBM5-Wnt/β-catenin in alveolarepithelial cells exposure to cigarette smoke PM2.5and to identifywhether RBM5is the upstream regulatory factor of Wnt/β-cateninsignaling pathway. This experiment also provides scientific basis forfurther study on the molecular mechanism of tobacco related lungdiseases, meanwhile, it provides new targets and perspectives for theprevention and treatment of pulmonary diseases.Method:Using0,10ug/ml,20ug/ml,40ug/ml,80ug/ml by cigarette smokeextract (CSE) or DMSO in24h,48h,72h gave the human alveolarepithelial cells (A549cells),the method comprises the following steps:(1) analysis of the survival rate of A549cells by MTT;(2) A549cell detection by Transwell invasion;(3) the expression level of RT-PCR, real-time-PCR detection ofRBM5mRNA, the expression level of Western; blot method to detect the RBM5and Wnt/β-catenin signaling pathway related components ofprotein;(4) the expression and distribution of immunofluorescent labelingcells in A549β-catenin:(5) flow cytometry, acridine orange staining and Western blot for thedetection of cell proliferation and apoptosis rate;(6) the transcription factor TCF Luciferase Report Gene Detection ofA549cell activity;(7) transfected by pcDNA3.1-RBM5/si-RBM5, using the Westernblot method was used to detect the expression of Wnt/-catenin signalingpathway related components of protein;(8) the blocking of the Wnt/-catenin signaling pathway by ICG-001,the expression of Western blot method to detect the RBM5and Wnt/-catenin signaling pathway related components of protein;Results:(1) CSE inhibits the proliferation of A549cells, with increasing CSEconcentration and action time, the growth inhibition rate of A549cells ismore big, has the time and dose dependence, when cigarette smokeextract concentration increased to80ug/ml,72h, A549cell death is more,suggesting that cigarette smoke extract can induce alveolar epithelial celldamage;(2) the expression of cell nucleus and cytoplasm of A549cellswithin the given CSE β-catenin protein increased, with the increase ofCSE concentration of β-catenin protein expression more, with dosedependent expression of A549cells; give the CSE detection ofWnt/β-catenin signal related components of the protein, the activation ofWnt signaling pathway components (Wnt2, Wnt7a, increased expressionof P-GSK3beta), inhibitory components (GSK3β, P-β-catenin) decreased expression of alveolar epithelial cell injury, suggesting that Wnt/β-cateninsignal pathway induced by cigarette smoke extract;(3) the detection of luciferase report gene to give CSE A549cellsshowed enhanced T cell transcription factor-TCF activity, suggestingthat Wnt/β-catenin signaling pathway activation of alveolar epithelial celldamage induced by cigarette smoke extract;(4) given CSE A549cells, RBM5mRNA increased with theincrease of CSE concentration and action time prolonged expressiongradually decreased, with time and dose dependence; with increasingconcentrations of cigarette smoke extract, reduce the expression of RBM5protein expression, with dose dependent, suggesting that RBM5wasinvolved in the extraction of alveolar epithelial cell injury caused bycigarette;(5) after pcDNA3.1-RBM5transfection, Wnt/β-catenin signalingpathway inhibition; siRBM5after transfection, Wnt/β-catenin signalingenhanced; give the Wnt signal pathway blocker ICG-001blockedWnt/β-catenin signaling pathway, the expression of RBM5and thedifference was not statistically significant.ConclusionThis study identifies that RBM5-Wnt/β-catenin signaling pathwayplays an important role in cigarette smoke PM2.5exposure inducedalveolar epithelial cell damage; this study also confirmed that RBM5isthe upstream negative regulatory factor of Wnt/beta-catenin signalingpathway;As the extension of cigarette smoke effect time and the increaseof the dose, the severity of injury increased from which we could inferpotential molecular mechanism that the amount of smoking and the yearof smoking aggregate lung injury.This study provides scientific basis forfurther study on the molecular mechanism of tobacco related lungdiseases.The main source of indoor cigarette smoke is PM2.5.Therefore, this study can also provide theoretical basis for mechanism of indoorPM2.5harm human health, meanwhile, it provides new ideas for themechanism of air pollution harm human health. |
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