Role Of MiR-378 In Paraquat-induced Apoptosis Of Type Ⅱ Alveolar Epithelial Cells

Objective: Paraquat(PQ),chemically known as 1,1 ’-dimethyl-4,4’-bipyridine cationic salt,is currently the most widely used organic heterocyclic contact herbicide in the world.The mortality rate of patients with acute oral PQ poisoning is very high because there is no specific detoxification drug.Lung is the main damaged organ of PQ poisoning,and pulmonary interstitial fibrosis is an important cause of death in patients with PQ poisoning.The excessive apoptosis of alveolar epithelial cells is considered to be one of the key events in the early development of pulmonary fibrosis.The apoptosis of alveolar epithelial cells induced by PQ involves many apoptotic pathways,among which the mitochondrial apoptotic pathway plays an important role.Other studies have shown that mitochondrial autophagy can inhibit cell apoptosis and increase the threshold of cell apoptosis,thus improving PQ-induced cell damage.Microribonucleic acids(miRNAs)are small,endogenous,non-coding ribonucleic acids that regulate the expression of target genes after transcription.The discovery of miRNAs has provided new insights into the regulatory mechanisms of multiple cellular processes,including autophagy and apoptosis.Mature micro RNAs are abbreviated as miR.Mi R-378 is a class of miRNAs that have been proved to be closely related to apoptosis in recent years.Studies have shown that miR-378 is closely related to the occurrence and development of many diseases,and the mechanisms involved are extremely complex.Taking the regulation of apoptosis of miR-378 as an example,the high expression of miR-378 in tumor cells can mostly promote the apoptosis of tumor cells,but there are also anti-apoptotic effects in other diseases,involving inhibition of caspase-3 and enhancement of mitochondrial autophagy.According to the studies on the high expression of miR-378 in nerve cells,skeletal muscle,cardiac muscle and other cells and tissues,we speculated that the high expression of miR-378 plays an important role in the process of apoptosis of Type II of alveolar epithelial cells(AEC2)during PQ injury,and it is planned to be confirmed in vitro cell experiments.Methods: 1.Detection of cell transfection efficiency of miR-378 mimics:(1)Cell transfection: MLE12 cells were transfected with miR-378 mimics and negative control;(2)Total RNA extraction and real-time quantitative RT-PCR were used to detect the expression of miR-378.2.Effects of miR-378 overexpression on PQ-induced apoptosis of MLE12 cells:(1)CCK-8 assay was used to detect cell activity;(2)The changes of nuclear morphology were observed by DAPI staining;(3)The apoptosis rate of cells in each group was detected by Annexin V-FITC/PI double-staining apoptosis kit;(4)The activity of caspase-3 /-9 was detected with the Caspases activity detection kit;3.Detection of indicators related to mitochondrial autophagy pathway in the process of PQ-induced apoptosis of Mle12 cells:(1)Mitochondrial autophagy was observed by fluorescence microscopy with MTG and LTR staining;(2)The expression levels of LC3 and β-actin were detected by Western Blotting.Results: 1.Detection of cell transfection efficiency: After transfection of miR-378 mimics into MLE12 cells,the expression level of miR-378 was significantly increased.2.Effects of miR-378 overexpression on cell activity: The cell viability of MLE12 cells transfected with miR378-mimics+PQ was significantly increased compared with that of MLE12 cells transfected with miR378-mimics+PQ.3.Effects of miR-378 overexpression on nuclear morphology and structure:Compared with the PQ group,the proportion of cells with nuclear pyknosis and fragmentation in the miR-378 mimics +PQ group was significantly lower.4.Effects of miR-378 overexpression on apoptosis rate: Compared with the PQ group,the apoptosis rate of miR-378 mimics +PQ group was significantly reduced.5.Effects of miR-378 overexpression on mitochondria-lysosome fusion:MLE12 cells were co-stained with MTG and LTR,and then analyzed by fluorescence microscopy.Compared with the PQ group,mitochondrial autophagy was significantly enhanced in the miR-378 mimics +PQ group.6.Effects of miR-378 overexpression on caspase-3 and 9 activities: The activities of caspase-3 and caspase-9 in PQ+miR-378 mimics were significantly lower than those in PQ group.7.Overexpression of miR-378 enhanced the expression of mitochondrial autophagy signature proteins: MLE12 cells by miR-378 transfection accept PQ again after processing,Western Blotting test shows that the mitochondrial autophagy related proteins LC3-Ⅱ / LC3-Ⅰ expression increases.Conclusion: In PQ-induced apoptosis of AEC2(MLE12)cells,overexpression of miR-378 may inhibit apoptosis by regulating the activity levels of mitochondrial autophagy,caspase-3 and 9.