The Role And Underlying Mechanisms Of MiR-582-3p On The Alteration Of Biological Behaviors Of A549 Cells Induced By Ambient PM_2.5

ObjectiveAmbient PM_2.5is one of the main environmental air pollutants.Ambient PM_2.5exposure is closely relevant to the development and progression of lung carcinoma,but the mechanisms remain unrevealed.In this study,the effects of ambient PM_2.5exposure on the malignant biological behaviors of A549 cells were explored in vitro,and the molecular mechanism was further explored.Methods1.Ambient PM_2.5was collected by the filter membrane method,weighed after fully drying and used the weight gain method to obtain the quality of PM_2.5.The PM_2.5 sample obtained after filter film cutting,ultrasonic and drying was dissolved inDMSO to obtain 100 mg/mL PM_2.5stock solution.2.A549 cells were treated with different concentrations of ambient PM_2.5,and the effects of PM_2.5on the proliferation,migration and invasion ability of A549 cells were detected by plate clone formation assay,wound healing assay and transwell cell invasion assay.The related proteins changes of EMT and Wnt/β-catenin signaling pathway were detected by western blot assay,the expression level of miR-582-3p was detected by q RT-PCR.3.Down-regulated the expression of miR-582-3p in A549 cells by transfected miR-582-3p inhibitor,and then treated the transfected cells with PM_2.5.The regulation effects of miR-582-3p on the proliferation,migration and invasion ability of A549 cells promoted by PM_2.5were detected by plate cloning formation assay,wound healing assay and transwell cell invasion assay,and the effects of miR-582-3p on EMT and Wnt/β-catenin signaling pathway activation in A549 cells induced by PM_2.5were detected by Western blot.4.To explore the regulatory mechanism,DKK-1 was used to inhibit the Wnt/β-catenin signaling pathway,and then detected the EMT of A549 cells by Western blot.ResultsAmbient PM_2.5induced biobehavioral alteration in A549 cells by activating Wnt/β-catenin signaling pathway1.The toxic effects of PM_2.5and DKK-1 on A549 cells were dose-dependent.The survival rate of A549 cells significantly decreased when PM_2.5was greater than 100 μg/mL（P<0.01）,and when DKK-1 was 50ng/mL,the toxicity to A549 cells was not different from that of the control group.Therefore,PM_2.5concentration was determined to be 0,25,50,100μg/mL,and DKK-1 intervention concentration was 50ng/mL.2.Ambient PM_2.5can cause the alteration of biological behaviors of A549 cells.Compared with the solvent control group,the ambient PM_2.5treatment significantly enhanced the proliferation,migration,and invasion ability of A549 cells.The strongest effect was when the PM_2.5concentration was 25μg/mL,and gradually decreased as the concentration increased.Ambient PM_2.5also promoted EMT of A549 cells,the expression of Vimentin,EMT marker protein,gradually increased with the concentration gradient of PM_2.5（0,6.25,12.5,25μg/mL）,while E-cadherin significantly decreased（P<0.05）.3.Ambient PM_2.5caused excessive activation of Wnt/β-catenin signaling pathway in A549 cells.With the increase concentration of PM_2.5,the expression level of β-catenin and P-GSK-3β/GSK-3β,Wnt/β-catenin pathway marker proteins,were significantly increased,while the expression level of APC was significantly decreased.The difference was statistically significant（P<0.05）.miR-582-3p mediated the process that ambient PM_2.5activated Wnt/β-catenin pathway and further caused the biobehavioral alteration of A549 cells1.Ambient PM_2.5caused the overexpression of miR-582-3p in A549 cells.2.After knocked down the expression of miR-582-3p in A549 cells,the proliferation,migration,and invasion of the cells were significantly decreased,and the activation of EMT and Wnt/β-catenin pathway were weakened.Furthermore,the above effects can be reversed by PM_2.5.3.After inhibited the Wnt/β-catenin signaling pathway,the EMT process of A549 cells was also inhibited,overexpression of miR-582-3p can reverse the inhibition of EMT process caused by Wnt/β-catenin pathway.ConclusionAmbient PM_2.5enhanced EMT of A549 cells by activated the Wnt/β-catenin signaling pathway,and then caused the proliferation,migration and invasion of cells accordingly.In addition,miR-582-3p was highly expressed in A549 cells after PM_2.5exposure and mediated the alteration of biological behaviors of A549 cells that caused by the activation of Wnt/β-catenin signaling pathway by PM_2.5.The molecular biological mechanism of PM_2.5promoted the development of lung cancer was explored in detail from the cellular level in vitro,which provided a new idea for further exploring the mechanism of the development of lung cancer caused by environmental pollution.