Dynamic Regulation Mechanism Of Let-7a-2-3p/PIK3CG Module On Apoptosis Level Of Macrophages Infected By Mycobacterium Tuberculosis

Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(M.tb).Despite thousands of years of human struggle against TB,10 million people still suffer from TB worldwide every year.The unclear pathogenesis and host immune mechanism of TB seriously hinder the development of diagnosis and treatment of TB.When the host is infected with M.tb,the macrophages,an important part of innate immune response,are activated quickly,becoming an important barrier for the host to resist the transmission of M.tb infection,and also becoming the main habitat and breeding place of M.tb.micro RNAs(mi RNAs),as epigenetic regulators of post-transcriptional regulation,are involved in many biological processes,especially in many aspects of pathogenic processes.Changes in the expression of mi RNAs in macrophages in response to M.tb infection can affect their huge target gene regulatory network,thereby regulating host immune response-related processes and pathways.Therefore,it is of great significance to systematically and thoroughly explore the expression changes of mi RNAs and their target genes in macrophage immune response to M.tb infection,to construct the regulation network about immune response of mi RNA-m RNA,and to explore the important functional regulation modules in host anti-infection process,so as to elucidate the pathogenic mechanism of M.tb and host anti-infection immune mechanism,and to provide new treatment for TB.The discovery of the method and the improvement of the level of clinical diagnosis lay a solid theoretical foundation.In this study,we established a model of macrophage infection by M.tb(H37Ra attenuated strain and H37 Rv standard strain).The expression profiles of mi RNAs and m RNAs in the model samples were measured by RNA-seq.The expression levels of mi RNAs and m RNAs were analyzed by bioinformatics method.The differentially expressed mi RNAs and m RNAs were fully excavated and the common differences between H37 Ra attenuated strain and H37 Rv standard strain were extracted.Differential mi RNAs and m RNAs were used to construct immune response regulatory network of macrophages infected with M.tb,and to further explore the significant molecular regulatory modules in host response to M.tb infection.At the same time,we identified the differentially expressed mi RNAs and m RNAs in peripheral blood mononuclear cells(PBMCs)of latent tuberculosis infection(LTBI)population,constructed a specific mi RNA-m RNA regulatory network related to LTBI.By comparing the mi RNA-m RNA regulatory network of M.tb in vitro infection of macrophages with the specific mi RNA-m RNA regulatory network in PBMCs of LTBI individuals,it was found that PI3K-Akt signaling pathway was significantly enriched in both networks.We identified the abnormal expression of let-7a-2-3p/PIK3 CG module in the regulation network of mi RNA-m RNA in vitro infection model as inhibiting the apoptosis of macrophages,promoting the secretion of macrophage proinflammatory factor-tumor necrosis factor-alpha(TNF-α),and enhancing the inflammatory response of macrophages to a certain extent.Part Ⅰ Establishment of a THP-1-derived macrophage model infected by M.tb and sequencing of the expression profiles of mi RNAs and m RNAsH37Ra attenuated strain and H37 Rv standard strain infected macrophages with a multiple infection of 10:1.Laser confocal microscopy showed that M.tb co-localized with lysosomes.ELISA results of supernatant of infected cells showed that the secretion of TNF-α increased with the prolongation of infection time.Both results indicated that the THP-1-derived macrophage model of M.tb infection was successfully established.The expression profiles of 9 samples(each group had 3 duplicate samples)from three groups(H37Ra attenuated strain infection group,H37 Rv standard strain infection group and control group)were obtained by RNA-seq,and the differential expression of mi RNAs and m RNAs were analyzed by bioinformatics tools.184 differentially expressed mi RNAs were screened in the H37 Ra attenuated strain infection group,among which 112 were up-regulated and 72 were down-regulated.There were 3938 differentially expressed genes in the H37 Ra attenuated strain infection group,of which 1457 were up-regulated and 2481 were down-regulated.There were 198 differentially expressed mi RNAs in the H37 Rv standard strain infection group,among which 102 were up-regulated and 96 were down-regulated.There were 4750 differentially expressed genes in H37 Rv standard strain,among which 1668 were up-regulated and 3082 were down-regulated.q RT-PCR was used to validate the differentially expressed profiles of mi RNAs and m RNAs between H37 Ra attenuated strain infection group and H37 Rv standard strain infection group.The results were consistent with the trend of sequencing data analysis,indicating that the differentially expressed mi RNA and m RNA profiles obtained by sequencing were relatively reliable and could be used for subsequent analysis.Part Ⅱ Analysis of the common characteristics of mi RNA-m RNA regulatory network in M.tb in vitro infection model and in PBMCs of clinical LTBI population54 differentially expressed mi RNAs and 3460 differentially expressed genes were screened out between H37 Ra attenuated strain infection group and H37 Rv standard strain infection group.Based on Target Scan and mi RDB target gene prediction database information,a mi RNA-m RNA regulatory network related to macrophage immune response in M.tb infection was established.The network consisted of 49 differentially expressed mi RNAs and 1414 differentially expressed genes.The abnormal expression of the genes in the regulatory network was further analyzed by DAVID online analysis tool.GO functional analysis showed that the target genes in the network were mainly annotated in the positive regulation of gene expression,activation of cysteine-type endopeptidase activity involved in apoptotic process,protein kinase binding and so on;pthway enrichment analysis showed that the target genes in the network were mainly concentrated in apoptosis,p53 signaling pathway,Cytokine-cytokine receptor interaction and other metabolic pathways,apoptosis pathway was the most significant.PI3K-Akt signaling pathway was the most prominent one among the many signaling pathways that make up cell apoptotic,and the target genes(PIK3CG,PIK3R3 and AKT3)in the regulatory network were enriched in this pathway.15 specifically expressed mi RNAs and 855 specifically expressed m RNAs were screened out from PBMCs of LTBI population and a specific mi RNA-m RNA regulatory network was established in PBMCs of LTBI individuals.The network consisted of 9 mi RNAs and 92 genes.Further PPI(protein-protein interaction)network analysis of the network target genes and pathway enrichment analysis revealed that the PI3K-Akt signaling pathway was the most significant,indicating that the PI3K-Akt signaling pathway may play an important role in the host’s response to M.tb infection.Part Ⅲ Dynamic regulation mechanism of let-7a-2-3p/PIK3 CG module on apoptotic level of macrophages after M.tb infectionPIK3CG is an important molecule of PI3K-Akt signaling pathway,and bioinformatics analysis shows that let-7a-2-3p and PIK3 CG have potential targeting regulation relationship.The expression levels of let-7a-2-3p and PIK3 CG in the model were verified by q RT-PCR.The results showed that let-7a-2-3p was down-regulated and PIK3 CG was up-regulated,which was consistent with the trend of RNA-seq data analysis.Western blot results showed that the expression of PIK3 CG protein product p110γ increased significantly in the infected group,indicating that PI3K-Akt signaling pathway was active in macrophages infected by M.tb,and the expression level of anti-apoptotic protein Bcl-2 was significantly increased.After overexpression of let-7a-2-3p,it was found that the expression of PIK3 CG and its protein product p110γ decreased.Luciferase reporter gene experiment confirmed that let-7a-2-3p could bind directly to the 1992-2013 or 3028-3049 loci of PIK3 CG 3’UTR region.Overexpression of let-7a-2-3p inhibited the secretion of pro-inflammatory factor TNF-α,and inhibited the expression of let-7a-2-3p,the secretion of TNF-α increased,and after knockdown of PIK3 CG gene,the secretion of TNF-α was inhibited.The abnormal expression of let-7a-2-3p/PIK3 CG module in macrophages infected with M.tb can promote the secretion of TNF-α.We found that the expression of Bcl-2 was significantly decreased by overexpression of let-7a-2-3p or knockdown of PIK3 CG gene in macrophages that was infectied with M.tb.CCK8 assay showed that the survival rate of macrophages decreased after M.tb infection,and the apoptotic rate of macrophages was significantly increased by flow cytometry,indicating that the expression chang of let-7a-2-3p/PIK3 CG module in macrophages,which had a certain impact on the apoptosis of macrophages after M.tb infection.let-7a-2-3p/PIK3 CG module showed abnormal down-regulation and up-regulation patterns respectively in macrophages infected by M.tb,which made PI3K-Akt signaling pathway in which PIK3 CG was located became active and futher promoted downstream Bcl-2 protein overexpression and inhibited macrophage apoptosis.This phenomenon also provided convenience for the retention of M.tb in macrophages.