Transcriptional Analysis Of Noncoding RNA In Macrophages Under Autophagic Stress And The Regulation Of Autophagy By Specific NcRNA

ResearchObjects Long noncoding RNA(Lnc RNA)regulation of life activities has become one of the hot issues in life-science research.Lnc RNA related research plays an important role in many areas of life sciences,such as stem cell biology,Developmental biology,neuroscience,immunology,and oncology.However,the transcriptional characteristics and functions of Lnc RNAs in specific cells,tissues,and disease models are not yet clear.At present,only a few Lnc RNAs have been revealed,and a large number of unknown Lnc RNAs are yet to be further discovered and identified.Macrophages are an important component of innate immunity.Fusion and acidification of macrophages autophagosomes with lysosomes are the most important ways to clear pathogens.Autophagy is regulated by many factors.Previous studies have shown that Lnc RNA is involved in the regulation of macrophage autophagy,but its detailed transcriptome pattern and the molecular mechanism of specific Lnc RNA-regulated autophagy remain unclear.Therefore,to analyze the macrophage autophagic stress-related Lnc RNA transcriptome profile,screen specific macrophages autophagy-specific Lnc RNAs,and investigate their molecular mechanisms,both to further reveal the molecular mechanism of macrophage autophagy,or to The role and mechanism of cognitive Lnc RNAs in macrophage pathogen immunity have important theoretical and practical values.In this study,macrophages as the research object,with a view to revealing under the conditions of autophagy,the transcriptome pattern of longchain non-coding RNA and the molecular mechanism of specific Lnc RNA molecules regulating cell autophagy.ResearchMethods First,the autophagic stress model of Rapa,autophagy induced by starvation,and autophagy inhibition by 3-MA were constructed.The transcriptome pattern of macrophage Lnc RNA was obtained by transcriptome sequencing analysis.Through functional annotation and network analysis,19 autophagy-related Lnc RNA-m RNA pairs were obtained.Eight significant differentially expressed Lnc RNA molecules were selected and confirmed by realtime fluorescence quantitative PCR(RT-q PCR).Secondly,in order to solve the technical difficulties of transient transfection of macrophage plasmids and to study the dynamic research on the effects of Lnc RNAs on the changes of macrophage autophagy,we constructed two-color fluorescent markers functional autophagy flux detection system based on the third-generation lentivirus system.Third,to determine whether specific Lnc RNA Malat1 regulates macrophage autophagy.By constructing a specific autophagy-related Ce RNA interaction network.The expression of Malat1 was inhibited by the lentivirus overexpression Malat1 or Smart Silence in the macrophage Raw264.7,and the autophagic flow was detected by the autophagic flowlentivirus system.The autophagic index proteins(ATG5,P62)were detected by Western Blot.And LC3 I/II)expression changes.Fourth,it is clear whether mir-23-3p in the regulatory axis of Malat1-mir-23-3p-Lamp1 can target autophagy of the lysosomal membrane-associated protein Lamp1.Bioinformatics analysis identified Lamp1 as a potential target of mir-23-3p.After transiently transfecting Raw264.7 with mimics and Inhibitor,endogenous LC3 was analyzed by immunofluorescence to detect the status and quantity of autophagosomes,autophagic flow changes were detected by confocal laser scanning,and Lamp1-3 UTR wildtype and mutant reporter plasmids were constructed.Dual luciferase reporter assays were used to determine the targeting relationship between the two.Mimics or Inhibitor gradient assays were used to detect luciferase activity and WB detection target Lamp1 and autophagy(ATG5,P62 and LC3 I/II)to determine whether mir-23-3p targets Lamp1-regulated macrophage autophagy.Finally,it was verified that Malat1 can regulate Lamp1-mediated autophagy as a mi RNA of mir-23-3p.The expression of Malat1-mir-23-3p and Lamp1 in Rapa model was detected by RT-q PCR.The Malat1-3 UTR wild-type and mutant reporter plasmids were constructed,and the dual luciferase reporter system was used to verify the interaction between Malat1 and mir-23-3p.The expression of Lamp1 m RNA was detected by RT-q PCR and the expression changes of LAMP1 and autophagy key proteins ATG5,P62,and LC3 were detected by Smart Silence technique,while Malat1 and mir-23b-3p were identified interaction and its effect on Lamp1 expression and autophagy.Main Results(1)Detailed Lnc RNA transcriptional expressions profile in macrophage were obtained.By RNA-SEQ and bioinformatics analysis,a large number of Lnc RNAs were found to respond to autophagic stress.After excavation,1127 Lnc RNAs were obtained,including 831 linc-RNAs,129 Introns,and 152 Antisense-Lnc RNAs.240 new Lnc RNAs were discovered.Lnc RNA differentially expressed 238.The average coding capacity and expression value of Lcn RNA was significantly lower than that of the coding gene and was evenly distributed on the chromosome with no preference.Autophagy GO and Pathway annotations obtained 83 and 104 Lnc RNA molecules related to autophagy.RT-q PCR analysis identified eight Lnc RNA molecules,including Malat1,AI662270,and GAS5,under Rapa-induced autophagy.The results were consistent with RNA-SEQ expression.(2)Successfully established a dual-label multi-functional autophagy flow detection and analysis technology system based on lentivirus.(3)Malat1 may promote macrophage autophagy with the Ce RNA mechanism.Autophagic Ce RNA network analysis showed that Malat1 can adsorb a large number of mi RNAs.RNAFISH showed that Malat1 is mainly distributed in the macrophage cytoplasmic region.Overexpression of Malat1 autophagy analysis confirmed that Malat1 promotes the fusion of autophagosomes with lysosomes and promotes the formation of autophagosomes.WB experiments proved that overexpression of Malat1 can promote the degradation of P62 and the conversion of LC3 I/II,while Malat1-(si RNA ASO)can reverse the above phenomenon.Therefore,Malat1 promotes macrophage autophagy.(4)Malat1 competitively adsorbs mir-23-3p to regulate autophagy.Dual luciferase reporter assay and WES experiments showed that Malat1 regulates macrophage autophagy by combining with mir-23-3p affecting Lamp1 expression,Malat1 can inhibit mir-23-3p activity through the Sponge role,and may release it to Lamp1,ATG12,Targeting silencing of PTEN,AKT1,etc.enhances the fusion of autophagosomes and lysosomes and promotes the occurrence of late autophagy.(5)Mir-23-3p targets Lamp1 to regulate macrophage autophagy.Dual luciferase reporter assays demonstrated that mir-23-3p targets the lysosomal membrane-associated protein Lamp1.Immunofluorescence analysis showed that overexpression of mir-23-3p could inhibit the formation of autophagosomes.Autophagic flow analysis showed that over-expression of mir-23-3p significantly inhibited macrophage autophagy.The detection of autophagic markers by WB after transfection of mimics and Inhibitor indicates that mir-23-3p can regulate macrophage autophagy.The above research methods and conclusions lay a solid foundation for further research on the interaction between Lnc-Malat1,mir-23-3p and Lamp1 in macrophage anti-pathogenic microorganisms.At the same time,a large number of non-coding RNAs identified by computational biology in macrophages and the establishment of research techniques are conducive to the advancement of long-chain noncoding RNAs in macrophages.