Molecular Mechanism Of MORN4 And NR4A2 On Mitochondrial Autophagy In A549 Cells

Research BackgroundFor damaged organelles and some unwanted substances in cells,cells can be eliminated by autophagy,thus providing material basis for cell metabolism and renewal of some organelles.Therefore,autophagy plays an important role in maintaining cell homeostasis.Autophagy includes non-selective autophagy and selective autophagy.Among them mitophagy is selective autophagy,and damaged mitochondria are cleared through autophagy pathway,so mitochondrial autophagy plays an important role in mitochondrial quality control.Sphingosylphosphorylcholine(SPC)is a biologically active sphingolipid that is naturally present in the blood and produced by sphingomyelin under the action of deacylase.Studies have found that SPC can regulate the proliferation,migration and apoptosis of cancer cells.Our previous laboratory studies found that SPC can promote autophagy in A549 cells and affect ROS and mitochondrial membrane potential.Mitochondrial membrane potential is reduced,accompanied by an increase in mitochondrial autophagy.At present,the role and mechanism of SPC in mitochondrial autophagy has not been clarified.For the analysis of gene chip results,we found and demonstrated that SPC can increase the expression of MORN4 and NR4A2.MORN4(the membrane occupation and recognition nexus repeats 4 times)is a protein comprising four MORN motifs.MORN motifs are widely found in proteins of animals,plants and protists,ranging from 2-17.Previous studies in the laboratory found that SPC up-regulated the expression of MORN4 in H9c2 cardiomyocytes,and found that SPC inhibited apoptosis by autophagy promoted by JNK/MORN4/MFN2.Whether MORN4 is involved in autophagy in A549 cells and whether it is capable of regulating mitochondrial autophagy is unclear.NR4A2(Nuclear receptor subfamily 4,group A,member 2)is also called Nurrl,and NR4A1,NR4A3 constitutes the NR4A orphan nuclear receptor family.The study found that NR4A2 plays a role in the development of tumors,obesity,especially neurological diseases.For example,in lung cancer cells,NR4A2 binds to p53 and inhibits apoptosis.The pre-laboratory phase also demonstrated that the ROS/NF-?B/NR4A2 pathway is involved in the process of hydrogen peroxide-induced apoptosis in mouse cardiac sca+ cells,but the role of NR4A2 in the SPC pathway has not been clarified.In this study,we found that after APC treatment,the expression of LC3-II increased,the mtDNA content decreased,the expression of Tim23 decreased,the mitochondrial membrane potential decreased,and the expression of mitochondrial synthesis-activated gene PGC1B increased.These results proved that SPC can promote A549 mitochondrial autophagy.It was further found that SPC can upregulate its expression.After using MORN4 lentivirus interference plasmid to reduce its expression,mitochondrial autophagy was further increased,indicating that MORN4 may play a negative feedback on the function of SPC.Since the expression of mitochondrial fusion protein mfn2 is decreased after the expression of MORN4 is decreased,it is speculated that MORN4 may affect mitochondrial autophagy by regulating mfn2.By analyzing the above results,we believe that SPC can induce mitochondrial autophagy in A549 cells,while mitochondrial autophagy leads to a decrease in mitochondria and an up-regulation of MORN4 and PGC1B expression.After up-regulation of MORN4,it causes the expression of downstream mfn2.mfn2 enhances mitochondrial fusion,frees part of mitochondria from autophagy to maintain the basal number of mitochondria,and apparently produces a decrease in autophagy.At the same time,after mitochondrial autophagy increased by SPC treatment or MORN4 interference inhibition,a new mitochondrial synthesis pathway including PGC1B was induced to increase the number of mitochondria and maintain the dynamic balance of mitochondria in cells.In the study of NR4A2,SPC also increased the expression of NR4A2,which was consistent with the results of gene chip detection;lentiviral transfection interfered with plasmid LV3-NR4A2,knocked down NR4A2,increased mitochondrial autophagy function of A549 cells,and study of MORN4 The result is similar,and this process may also be implemented by mfn2;After overexpression of NR4A2,autophagy increased and mitochondrial autophagy decreased in A549 cells.Overexpression of NR4A2 and SPC.treatment increased LC3-? levels,increased mtDNA content and Tim23 expression,and decreased PGC1B content,indicating that NR4A2 is involved in SPC-promoting mitochondrial autophagy in A549 cells.