Effects Of Let-7d In SiO₂-induced Epithelial-mesenchymal Transition Of Alveolar Epithelial Cells

Background and objectiveSilicosis is one of the most serious diseases in pneumoconiosis,and it is also one of the difficult points in the prevention and treatment of occupational diseases.At present,it is believed that during the pathogenesis of silicosis,the proliferation of myofibroblasts and the secretion of related proteins are the key links in the fibrosis stage in which the silicosis is irreversible from the inflammatory stage.The epithelial-mesenchymal transition?EMT?of pulmonary epithelial cells,as an important source of myofibroblasts,has attracted much attention.Previous studies have suggested that microRNA let-7d and its target gene high mobility group AT-hook 2?HMGA2?participate in the EMT process of cancer and renal fibrosis,but its role in the silicosis-EMT process has not been reported.Therefore,this study intends to use Transwell to construct a co-culture model of macrophages and pulmonary epithelial cells to imitate the EMT process in vitro,and observe the role of let-7d and HMGA2 in the process of SiO₂-induced EMT.Then the method of liposome transfection was used,and the expression of HMGA2 and other EMT-related genes were detected.Through the above research,we could find the related intervention effects and mechanisms,in order to provide new ideas for the study of SiO₂-induced EMT.Methods1.The establishment and observation of SiO₂-induced EMT model in vitroTHP-1 cells were induced to differentiate into macrophages by 100ng/ml PMA.The SiO2 concentration of THP-1-induced macrophages was determined by CCK-8assay.The co-culture EMT module was established by Transwell chamber.The experiment was divided into a blank control group and SiO₂ exposure group.The expression of epithelial marker E-cad,interstitial markers?-SMA and VIM were detected after SiO₂ stimulation,and the expression of let-7d and HMGA2 were also detected.2.The effects of silencing HMGA2 on SiO₂-induced EMT model in vitroThe expression of HMGA2 in A549 cells was specifically silenced by siRNA transfection,and the siRNA sequence with the best silencing efficiency was selected by qPCR.The experiment was divided into negative control group,SiO₂ exposure group,siR-HMGA2 group,and siR-HMGA2+SiO₂ exposure group.The changes of E-cad,?-SMA,VIM,and HMGA2 were detected by qPCR and WB after SiO₂ stimulation.3.The effect of intervention let-7d expression on SiO₂-induced EMT model in vitroThe Dual Luciferase Reporter Assay was used to verify that HMGA2 is the target of let-7d.Then let-7d mimics and let-7d inhibitor were transfected in the SiO2-induced in vitro EMT co-culture model to intervene in the expression of let-7d in A549 cells.The experiment was divided into negative control group,SiO₂ exposure group,let-7d mimics/let-7d inhibitor group and let-7d mimics/let-7d inhibitor+SiO₂ exposure group.qPCR and WB were used to detect E-cad,?-SMA,VIM and HMGA2 changes after SiO₂ stimulation.4.Statistical analysisThe experimental data were analyzed using SPSS21.0 software.The results under normal distribution were expressed as the mean±standard deviation,t-test was used when comparing the differences between any two groups.The differences between multiple samples were analyzed by ANOVA.Inspection level of alpha=0.05.Results1.The identification and detection of SiO₂-induced EMT model in vitroCCK-8 method was used to detect the viability of THP-1-induced macrophages exposed to SiO₂.When the concentration of SiO₂ was 150?g/ml,the cell survival rate was close to 50%.Compared with the control group,the expression of E-cad was decreased and the expression of?-SMA and VIM was increased in the SiO₂ group.At the same time,the expression of let-7d was decreased and the expression of HMGA2was increased.The difference was statistically significant?P<0.05?.2.The expression of EMT-related indicators after silencing the HMGA2 geneBoth transfection reagent and siR-NC could not affect the expression of HMGA2.The mRNA and protein expression of HMGA2 was decreased in A549 cells transfected with siR1-3.The silencing efficiency of siR-3 was the highest,and the difference was statistically significant?P<0.05?.Comparing with the negative control group,E-cad,?-SMA,VIM,and HMGA2all had significant differences?P<0.05?in mRNA and protein of A549 cells in the siR-HMGA2 group,the same as the siR-HMGA2+SiO₂ exposure group compared with the SiO2 exposure group.3.The expression of EMT-related indicators after the intervention of let-7dThe result of Dual Luciferase Reporter Assay revealed that co-transfection of let-7d mimics and mimics NC with the HMGA2 3'-UTR of wild type could significantly reduce the luciferase activity level at?82.08±0.61?%.But co-transfection of let-7d mimics and mimics NC with the mutated HMGA2 3'-UTR failed to diminish the luciferase activity level when compared with its control?P>0.05?.Transfection of let-7d mimics significantly up-regulated the expression of let-7d in cells.After let-7d expression was up-regulated,HMGA2 expression was reduced.Comparing with the negative control group,E-cad,?-SMA,VIM,and HMGA2 all had significant differences?P<0.05?in mRNA and protein of A549 cells in the let-7d mimics group,the same as the let-7d mimics+SiO₂ exposure group compared with the SiO₂ exposure group.Transfection of let-7d inhibitor significantly down-regulated the expression of let-7d in cells.After let-7d expression was down-regulated,HMGA2 expression was increased.Comparing with the negative control group,E-cad,?-SMA,VIM,and HMGA2 all had significant differences?P<0.05?in mRNA and protein of A549 cells in the let-7d inhibitor group,the same as the let-7d inhibitor+SiO₂ exposure group compared with the SiO₂ exposure group.Conclusions1.SiO₂ could induce EMT in alveolar epithelial cells in vitro,in which the expression of let-7d was decreased and the expression of HMGA2 was increased.2.By interfering with the expression of let-7d and HMGA2,the EMT process of SiO2-induced alveolar epithelial cells in vitro could be affected.