MiR-144-3p Targeted Regulation Of ATG4a Mediated BCGsurvivals By Inhib It Autophagy Formation And Autophagy-lysosome Mature

Objective Aims to explore miR-144-3p targeted regulation of ATG4 a mediated BCG survivals by inhibit autophagy formation and autophagy-lysosome mature.Method(1)Real-time PCR to detect the expression levels of miR-144-3p within BCG infection of RAW264.7 cells,and then use bioinformatics analysis software to predict its targeted genes.(2)Build pMIR-Report-ATG4a/mut-3 ’UTR recombinant plasmid.Using Dual-luciferase Report system,Real Time PCR and Western blot to detect luciferase activity,ATG4 a mRNA and protein expression level,verify targeted regulation of relations with ATG4 a of miR-144-3p.(3)By transmission electron microscopy,laser confocal microscope,Real-Time PCR and Western blot method to observe the autophagosome and autophagy-lysosome ultrastructure,phagosome formation and the distribution of mature and autophagy-lysosome,ATG4 a,P62 and the expression of LC3II/I,to research the mechanism of miR-144-3p on cell autophagic formation and the mature of autophagy-lysosome.(4)miR-144-3p mimic,inhibitor transfection into RAW264.7 macrophages,respectively,or with rapamycin,3-methyl adenine(3-MA)stimulation RAW264.7 macrophages,on the basis of using BCG(MOI=10)infected cells after 24 h,using the method of Real-Time PCR detection of BCG in each cell survival,in this study,aim to reveal the effects of miR-144-3p on the survival of BCG..Results(1)BCG infected macrophage model in different time show that in 12 h,24 h theexpression of miR-144-3p levels significantly increased(p < 0.01),with the infected of 48 h,miR-144-3p was lower than before,but still higher than uninfected group(p < 0.05).(2)Transfection miR-144-3p minic group,Atg4 a relative luciferase activity lower than the mutant 5.5 times(p < 0.05),and 6.1 times lower than normal control group(p< 0.05);but transfection miR-144-3p inhibitor group,ATGT4 a luciferase activity was increased,about 6.6times(p< 0.05);Through the Real-time PCR and Western Blot indicated that miR-144-3p can combine the 3 ’UTR target of autophagy related gene Atg4 a to inhibit its expression.(3)RAW264.7 cells with miR-144-3p mimic can inhibiting the formation of acid lysosome and autophagosome;on the contrary,after joining miR-144-3p inhibitor,intracellular punctiform autophagosome and autophagy-lysosome significantly increased,and crowded around the cell membrane;at the same time,miR-144-3p overexpression can make the protein level of ATG4 a and LC3II/I ratio decreased significantly,accumulation of P62 protein was higher than normal group;inhibited the miR-144-3p expression,the expression of ATG4 a and LC3II/I was higher than that of control group,and compared with the effect of BCG macrophage 24h(p<0.05),P62 protein content was lower than that of normal cells group.(4)The content of fluorescence quantitative PCR results show that detection of RAW264.7 BCG in cells,transfection of miR-144-3p plasmid survival capacity of BCG is higher than the normal group(p<0.01);on the contrary,inhibit the expression of miR-144-3p,BCG were significantly lower than the survival of normal group.Conclusion Through this experiment research proves that expression of miR-144-3p can be targeted autophagy related gene ATG4 a expression,inhibition of autophagosome and autophagy-lysosome mature,thus benefit to mycobacterium tuberculosis survive in macrophages.