| Background:Mycobacterium tuberculosis(Mtb)is a pathogen that causes tuberculosis in humans and animals.Macrophages are the main host cells of Mtb,which can clear intracellular Mtb through autophagy and limit its growth,however,Mtb have developed strategies to escape from the clearance.Numbers of studies have shown that long non-coding RNAs(LncRNAs)can be involved in the regulation of autophagy in many diseases.Among them,LncRNA-Cox2 is one of the most studied LncRNAs.However,the role of LncRNA-Cox2 in regulating macrophage autophagy during Mtb infection remains unclear.Therefore,it is of significance to explore the function of LncRNAs in regulating autophagy during macrophage defense process.Purpose and methods:In this study,si-RNA transfection was applied to establish LncRNA-Cox2 stably interfred raw264.7 cell strains.GFP fluorescent labeling and RealTime-PCR determined the transfection efficiency and interference efficiency of si-RNA.Using BCG induction to establish a macrophage infection model to study the effect of interfering LncRNA-Cox2 on the autophagy process of macrophages.Transmission electron microscopy,m-RFP-GFP-LC3 double fluorescent labeling protein,flow cytometry,RT-PCR and Western Blotting were used to investigate the regulation of autophagy in murine alveolar macrophages infected with BCG from cell morphological levels and cellular levels respectively.Further studies with MHY1485(The specific agonist of mTOR pathway)focus on discussing Mtb-induced autophagy regulated by LncRNA-Cox2 through the mTOR-dependent pathway.The results are as following:(1)BCG infection significantly increased the expression of LncRNA-Cox2.We established LncRNA-Cox2 interfered RAW264.7 cell strains by using si-RNA interference and the expression levels of autophagy-related proteins LC3Ⅱ,LAMP2,and Beclinl were initially detected.The results showed that interference with LncRNA-Cox2 could promote the expression of proteins such as LC3Ⅱ,LAMP2 and Beclinl,and did not affect the expression of macrophage surface marker antigen F4/80 and cell survival rate which provided a good experimental material for subsequent investigation of regulation of LncRNA-Cox2 in BCG-induced macrophage autophagy.(2)After BCG infection,interference with LncRNA-Cox2 was significantly up-regulated the expression of autophagy-related proteins LC3 Ⅱ,Atg5,Atgl2,and Beclinl and the number of intracellular autophagosomes and autophagolysosomes.This demonstrated that interference with LncRNA-Cox2 promoted the formation of intracellular autophagosomes,which may play a positive regulatory role in BCG infection-induced macrophage autophagy.(3)After BCG infection,interference with LncRNA-Cox2 inhibited the expression of p-mTOR and its downstream key factors p-S6 and p-ULK1(S757),however,down-regulate the expression of p38-MAPK and p-PI3K,and significantly up-regulate the expression of AMPK-a,while p-AKT and JNK did not change significantly.Activation of mTOR signaling with MHY 1485 can inhibit macrophage autophagy flow levels,up-regulated the expression of autophagy-related proteins LC3Ⅱand p-S6 in BCG-infected cells,while Atg7 was significantly down-regulated,and Beclin1 and p62 did not change significantly.The above results indicate that the regulation of autophagy by LncRNA-Cox2 is related to the mTOR-dependent pathway.Conclusion:In summary,LncRNA-Cox2 is up-regulated to participate in the regulation of autophagy during the process of Bacillus Calmette-Guerin infection-induced macrophages.Interfering with LncRNA-Cox2 inhi-bits mTOR-dependent pathway squamous acidification,inhibits mTOR signal transduction pathway activity,further up-regulates the expression of autophagy-related regulators,promotes autophagosome formation.It is speculated that down-regulated LncRNA-Cox2 is involved in the regulation of BCG-induced macrophages RAW264.7 autophagy may be through inhi-bition of mTOR signaling pathway,promotion of LC3 Ⅱ pression,resulting in blocked autophagosome membrane formation,and then play a positive regulatory role in autophagy. |
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