Study On The Effects Of MiR-96 Targeted FOXO3a At The Cytotoxicity Induced By Nanoscale Carbon Black Particles In 16HBE Cells

Objective:To investigate the toxicity induced by CB in 16HBE cells and the effects of miR-96 targeted FOXO3a.Methods:16HBE cells were cultured in MEM medium containing 10%heat inactivated fetal bovine serum at 37℃with a 5%CO₂ saturated humidity.The 16HBE cells in logarithmic growth phase were exposed to CB for 24 hours at a dose of 0,50,100 and 200μg/ml and MEM（containing 0.04%Tween80）as a negative control.RT-qPCR was used to detect miR-96 levels.The ROS levels were detected by flow cytometry.The rate of apoptosis was determined by flow cytometry staining with AnnexinV-FITC and PI.DNA damages were detected by Single Cell Gel Electrophoresis（Comet Assay）.The expression of FOXO3a protein was determined by Western Blot.The specific miR-96 Inhibitor and miR-96 Mimic were designed.The Lipofectamine^TM 2000 were used to transiently transfect with miR-96 Mimic and Inhibitor to construct the over expression and silence of miR-96 in 16HBE cells.Result:Compared with the control,after 100μg/ml CB treatment,the adherent ability and the refractive index of the 16HBE cells partially decreased;the ROS levels,the OTM value,and the Ratio of Apoptosis significantly increased.The expression of FOXO3a protein increased to 1.6-folds.Rather,the miR-96 levels significantly decreased.Transfected with mi R-96 Mimic transiently,the mi R-96 levels increased to 7.01-folds,whereas transiently transfected with miR-96 Inhibitor,the mi R-96 levels fell to 47 percent,manifesting both mi R-96 over expression cell line and miR-96 silence cell line were successfully constructed.The ROS level,OTM value,the Ratio of Early Apoptosis,the Ratio of Late Apoptosis,and FOXO3a protein were significantly increased while the Ratio of Survival was significantly decreased in the NC Mimic group,Mimic group,NC Inhibitor group and Inhibitor group after exposed to CB.After 100μg/ml CB treatment,compared with the NC Mimic group,the ROS levels,the OTM value,the Ratio of Early Apoptosis,the Ratio of Late Apoptosis,the Ratio of Survival,and FOXO3a protein levels increased to 0.78-,0.51-,0.70-,0.49-,1.05-,0.67-folds in the Mimic group,respectively;Namely after CB treatment,the increase of each index of the Mimic group was lower than that of NC Mimic group exposed to CB.while after 100μg/ml CB treatment,compared with the NC Inhibitor group,the ROS levels,the OTM value,the Ratio of Early Apoptosis,the Ratio of Late Apoptosis,the Ratio of Survival,and FOXO3a protein levels respectively increased to 1.57-,2.84-,1.99-,1.63-,0.89-,1.72-folds in the Inhibitor group;Namely after CB treatment,the increase of each index of the Inhibitor group was higher than that of the NC Inhibitor group exposed to CB.Conclusion:CB could induce the increases of ROS,DNA damages and cell Apoptosis in 16HBE cells.The miR-96 levels decreased after CB treatment.Over expression of miR-96 could decrease the ROS,DNA damages and cell Apoptosis induced by CB in 16HBE cells whereas miR-96 silence elevated the ROS,DNA damages and cell Apoptosis.The increased expression of FOXO3a protein in 16HBE cells after CB treatment,which might relate with the decreases of miR-96 levels induced by CB.