Bioinformatics Analysis Of Differential Expression Profiles Of MiRNA In Autophagy Model And MiR-129-3p Favors Intracellular BCG Survival By Inhibiting Autophagy

Objective To identify new miRNA that related to autophagy by bioinformatics technology,and explores the mechanism of miR-129-3p regulating autophagy by targeting Atg4 b.And reveal mechanism ofimmune escaping of Mycobacterium tuberculosis.Methods The Autophagy models was constructed by rapamycin or 3-MA,and the differential expression profiles of miRNA were obtained by high throughput sequencing technology,and the functional GO annotation,KEGG pathway analysis and protein interaction of miR-129-3p and its target genes were made by using miRWalk 3.0,DAVID,STRING and other databases.RT-qPCR was used to detect the expression level of miR-129-3p in autophagy or BCG infection model.The pMIR-Report-Atg4b-WT/MUT vector was constructed,and the targeting regulation of miR-129-3p on Atg4 b was verified by the methods of luciferase reporter experiment,RT-qPCR,Western Blot and so on.Western Blot was used to detect the expression of autophagic marker protein LC3 and p62.The distribution of fluorescence labeled LC3 in the cytoplasm was detected by laser confocal microscopy.The CFU assay was used to detect the survival of BCG.Results 1.Bioinformatics analysis showed that miR-129-3p was highly associated with autophagy.Atg4 b,a target gene of miR-129-3p,has many functions related to autophagy,and is involved in multiple autophagy-related signaling pathways.It plays an important role in the regulation of autophagy.2.RAW264.7 cellswere infected by BCG and the expression of miR-129-3p increased along with the increase of MOI,suggesting that miR-129-3p was associated with the degree of BCG infection.At different time points,the expression of mi R-129-3p increased gradually,and reaching the peak at 12 hours and then decreasing.It shows that miR-129-3p plays a role in the early stage of Mycobacterium tuberculosis infection and is subsequently regulated by certain substances in the cell.3.Dual-luciferase assay results showed that miR-129-3p mimic could significantly inhibit luciferase activity in pMIR-Report-Atb4b-WTgroup,and miR-129-3pantagomir could enhance luciferaseactivity.In the pMIR-Report-Atb4b-Mut group,the activity of luciferase was not changed with miR-129-3p mimic of antagomir exists.It suggested that miR-129-3p can interact with the 3 ’non-translation region of Atg4 b and inhibit its expression.At the same time,qRT-PCR showed that miR-129-3p mimic Atg4 b can inhibit the expression of mRNA,antagomir can increase the expression of mRNA Atg4b;Western Blot showed that miR-129-3p mimic can Atg4 b protein expression,miR-129-3p antagomir can increase the expression of Atg4 b.This suggests that miR-129-3p can regulate the expression of Atg4 b at the transcriptional level target.4.Western Blot datademonstrated that overexpression of miR-129-3pcould inhibit Atg4 b and LC3 I expression,and p62 protein aggregation was increased;miR-129-3p antagomir could increase Atg4 b,LC3I and LC3 II expression,and amplify the degradation of the Atg4 b protein,resulting in the decrease of its expression.The results of immunefluorescence showed that LC3 was distributed in the cytoplasm when cell transfected with mi-129-3p mimic and miR-129-3p antagomir could increase the puncta of LC3.It is suggested that miR-129-3p inhibits the formation of autophagosome and inhibits the level of autophagy.5.CFU results showed that miR-129-3p mimic could increase survival of intracellularBCG,on the contrary,miR-129-3p antagomir could reduce BCG intracellular survival but not as much as rapamycin do.Conclusion A new miRNA: miR-129-3p that related to autophagy was found in this experiment.It is proved that miR-129-3p can inhibit autophagy related protein Atg4 b and regulate autophagosome formationfurther affect the level of autophagy.MiR-129-3p can promote the survival of intracellular BCG,and play an important role in mechanism of MTB latent infection and immune evasion.