Study On Effect Of MiR-29a-3p On Macrophages Resistance To Mycobacterium Tuberculosis Infection

Objective:Tuberculosis(TB)is one of respiratory infectious disease that damages human health.The pathogenic bacteria-Mycobacterium tuberculosis(MTB)infection the lungs are engulfed and recognitioned by the first line of host defense phagocytic cells macrophages.Mycobacterium tuberculosis can regulate such as inhibiting apoptosis,inhibiting phagosome-lysosome fusion,inhibiting interfering signal transduction,inhibiting cytotoxic actions of ROS and RNS and inhibiting autophagy take part in the host immune response in Mycobacterium tuberculosis infection.The expression of miR-29a-3p was increased after infection with Mycobacterium tuberculosis.In this study,to observe the changes of apoptosis after transfected with pEZX-AM02-miR-29a-3p vector in RAW264.7,we aimed to explore the functional mechanism of miR-29a-3p in macrophages RAW264.7 in response to mycobacterial infection,to develop the new approach to diagnosis and treatment of TB.Method:This study through the miR-29a-3p target genes prediction by Target Scan?miRDB?PicTar and related bioinformatics software analysis by DAVID.The caspase3,caspase7,caspase8,Bcl-2,Mcl-1 were predicted as potential target genes of miR-29a-3p in apoptosis signaling pathway.After macrophage RAW264.7 cells were transfected with pEZX-AM02-miR-29a-3p vector or miR-29a-3p Nc control vector at 24,36,48 h,the expression change of miR-29a-3p,caspase3,caspase7,caspase8,Bcl-2,Mcl-1,Bax was detcted by real-time PCR,compare the change of apoptosis rate by means of flow cytometry and analysis the activity of caspase-3 and caspase-8 by measure absorbency.To search for the target gene in the functional mechanism of miR29a-3p in macrophages RAW264.7 in response to mycobacterial infection.Result:1.The miRNA inhibitor control and pEZX-AM02-miR-29a-3p expression vector were obtained.And optimization of the transfection conditions,When the ratio of plasmid and transfection reagent was 1:5,after RAW264.7 cells were transfected with pEZX-AM02-miR-29a-3p vector at 6h,replaced with completed medium,the transfection efficien was the best.2.After macrophage RAW264.7 cells were transfected with pEZX-AM02-miR-29a-3p vector,the expression of miR-29a-3p was decreased.3.After macrophage RAW264.7 cells were transfected with pEZX-AM02-miR-29a-3p vector,the expression of caspase3,caspase7,caspase8,Bcl-2,Mcl-1 and Bax are increaseed in different levels,and promoted apoptosis.4.By means of flow cytometry we find that after macrophage RAW264.7 cells were transfected with pEZX-AM02-miR-29a-3p vector,the rate of apoptosis was increased compared with the control group,The rate of apoptosis is the highest at 36h(P<0.05).5.After macrophage RAW264.7 cells were transfected with pEZX-AM02-miR-29a-3p vector,the activity of caspase-3 and caspase-8 increased not significantly.Conclusion: The apoptosis was increased after inhibition of miR-29a-3p expression by regulating the target genes caspase3,caspase7,caspase8,Bcl-2,Mcl-1and Bax.The functional mechanism of miR-29a-3p in macrophages RAW264.7 in response to mycobacterial infection is an inhibition of apoptosis to escape from the immune killing.