Analysis Of Changes In MicroRNA Expression Profile In PQ-induced Injury Of Mouse Alveolar Epithelial Cells

Objective: Paraquat(PQ),known as 1,1′-dimethyl-4-4′-bipyridinium dichloride chemically,is a highly effective herbicide.Because of its high herbicidal effectiveness and rapid degradation in soil,it is used in agricultural production worldwide.However,PQ is highly toxic to humans and rodents,and even a very small amount of PQ can lead to fatal consequences.Since the first report of death caused by PQ poisoning in the 1960 s,poisoning cases caused by taking or accidental contact with PQ often appear all over the world,especially in developing countries.Because the mechanism of PQ poisoning is not perfect,and the lack of specific antidotes,the mortality of patients with PQ poisoning remains high,so the treatment of PQ poisoning has become a thorny medical problem.Therefore,further revealing the molecular mechanism of PQ induced injury of alveolar epithelial cells will help to provide theoretical basis and possible therapeutic targets for the treatment of PQ poisoning.Micro RNA(miRNA)is a kind of highly conserved non coding RNA with a length of about 18 to 24 nucleotides.By binding with 3 ’-untranslated region(3’-UTR),it regulates cell proliferation and differentiation,signal transduction,apoptosis,tumorigenesis and development and other physiological and pathological activities.In recent years,miRNAs have been used to regulate gene expression in various biological and pathological processes,and has become an important field in biomedical research.PQ can induce alveolar epithelial cell injury through a variety of mechanisms,including lipid peroxidation caused by oxidative stress,inactivation of protein cross-linking,mitochondrial and chromosomal injury,acute inflammatory response,and injury of pulmonary micro-vascular barrier.In this process,the level of miRNA which is regulator of gene expression is likely to change,so that play a role in promoting or inhibiting injury.Although there have been some reports about the relationship between miRNAs and PQ poisoning,there are few studies on the miRNA expression profile in the process of PQ induced lung epithelial cell injury,and the regulatory mechanism of these miRNAs remains to be clarified.In this study,a mouse lung epithelial cell line(MLE-12)was selected as an in vitro model to investigate PQ induced lung epithelial cell injury,and the related indexes were observed,and the changes of miRNA expression profile were detected.Furthermore,the relationship between the genes regulated by differentially expressed miRNAs and the related signaling pathways and the injury of alveolar epithelial cells was further explored.Methods: 1.Cell viability test of MLE-12 cell injury model induced by PQ:(1)MLE-12 cells were cultured and treated with different concentrations of PQ;(2)Cell viability was detected by CCK-8 assay.2.Detection of apoptosis rate in MLE-12 cell injury model induced by PQ:(1)MLE-12 cells were cultured and treated with different concentrations of PQ;(2)The apoptosis rate was detected by FITC / PI double staining test kit.3.Microarray detection of miRNA expression profile changes and verification of differentially expressed miRNAs after PQ induced MLE-12 cell injury for 24 hours:(1)MLE-12 cells were cultured and treated with PQ;(2)Total RNA was extracted by TRIzol following the instruction;(3)Using Agilent mouse miRNA(8 * 60K)v21.0 to detect miRNAs;(4)Agilent scanning control software is used for scanning,and Agilent feature extraction software v10.7.1.1 is used for data extraction;(5)MLE-12 cells were cultured and treated with PQ;(6)Total RNA was extracted by following the instruction;(7)RT-PCR was used to detect the expression of miRNAs.4.Target gene prediction of differentially expressed miRNAs and statistical analysis of target gene results:(1)Using mi RDB and Target Scan to predict the target genes of differentially expressed miRNAs;(2)GO and KEGG were used to enrichment analysis of the target genes;(3)The miRNAs m RNA network was constructed by using Cytoscape 3.61,and the relationship between them was analyzed.Results: 1.PQ induced MLE cell injury and decreased cell viability:(1)PQ showed obvious cytotoxicity in MLE-12 cells,and the cell viability was time and concentration depended reduced;(2)When the treatment time was 24 h and PQ concentration was100μM,the cell viability was about half of the original.2.PQ attenuates PQ induced apoptosis of MLE-12 cells through mitochondrial apoptosis pathway: PQ could induce apoptosis of MLE-12 cells in a concentration dependent manner.Change of miRNA expression profile in PQ induced MLE-12 cell injury and verification:(1)In PQ induced MLE-12 cell injury,the following miRNAs were significantly down regulated:mmu-mi R-106b-5p,mmu-mi R-125a-5p,mmu-mi R-130a-3p,mmu-mi R-19b-3p,mmu-mi R-23a-3p,mmu-mi R-23b-3p,mmu-mi R-24-3p,mmu-mi R-25-3p,mmu-mi R-26a-5p,mmu-mi R-29a-3p;the following miRNAs were significantly up-regulated: mmu-mi R-1892;(2)The down-regulation of mmu-mi R-106b-5p,mmu-mi R-125a-5p,mmu-mi R-130a-3p,mmu-mi R-19b-3p,mmu-mi R-23b-3p and mmu-mi R-29a-3p was consistent with the results of microarray.4.Prediction of target genes of differentially expressed miRNAs and statistical analysis of target gene results: A total of 4183 genes may be the target genes of these differentially expressed miRNAs,of which 633 genes can be regulated by a variety of miRNAs.(1)The results of Gene Ontology analysis showed that the differentially expressed miRNAs mainly affected the mitochondrial apoptosis pathway(positive regulation of mitochondrial membrane protein insertion and apoptosis signal transduction,positive regulation of endogenous apoptosis signal transduction by osmotic stress)and DNA methylation(Synthesis and catabolism of 5-methylcytosine);(2)KEGG pathway analysis showed that the target genes of differentially expressed miRNAs were closely related to PI3 K Akt signaling pathway,m TOR signaling pathway,RAS signaling pathway and TNF signaling pathway.Conclusion: 1.When MLE-12 cells were injured by PQ,the cell viability decreased in a concentration dependent manner and the apoptosis rate increased in a concentration dependent manner.2.When PQ induced MLE-12 cell injury,the miRNA expression profile changed,10 miRNAs were significantly down regulated,and 1 miRNA was significantly up-regulated.The results of fluorescence quantitative PCR verified the accuracy of the microarray.3.The prediction of miRNAs’ target genes and result enrichment analysis suggest that these differentially expressed miRNAs may be closely related to mitochondrial apoptosis pathway and DNA methylation process.