Study On Rna Commumication From Host Macropage Into Invaded Mtb/bcg

Tuberculosis caused by Mycobacterium tuberculosis complex(MTBC)is a category of chronic zoonosis,which is regarded as one of the most serious infection disease worldwide.MTB,a facultative intracellular bacterial parasite,is primarily devoured by macrophages after infection,and the least part of bacteria escaped the killing system will entrenched in macrophages long-term.MTB has coevolved with host macrophages for thousands of years and sophisticated interaction mechanisms are involved between them.Although numerous published studies have indicated the interaction mechanisms between MTB and macrophages from various levels including organism,cell and molecule,there is limited information on how MTB escape killing system in macrophages.Given this limited information,this study was carry out to evaluate MTB and macrophages interaction in respect of transcriptome via an accurate and efficient method provided by RNA-seq high-throughout sequencing technology.Non-coding RNA and mRNA used in RNA-seq were extracted from THP-1 cell infected with MTB/BCG.The transcriptomes of interaction between the MTB/BCG and macrophages were compared by bioinformatics analysis.Our results reveal that there is cross-species translocation of RNA of host macrophage into MTB/BCGThe main contents are as follows:1.Bioinformatics analysis of high-throughout deep sequencing data.To ensure high quality,this study took mapped reads in annotated gene regions as a standard for the high-throughput deep sequencing data and conduct a comprehensive and credible analysis.The gene expression of 6 THP-1,4 MTB and 4 BCG samples reached76.43%,97.79%and 98.18%of genes of their genome,respectively.The gene expression of these sample met the requirement of establishing database since detected genes were saturated in RNA-seq.This research also investigated located sequence numbers and their rate in effective reads in the sequencing.84%?92%of effective sequences of THP-1 cells enabled to map to human's reference genome.No significant difference of transcriptomewas found between infectedTHP-1 cells and their uninfected control.6.25%-45.97%of RNA-seq sequences of MTB extracted from intracellular were mapped to the reference strain H37RV,however,49.29%-67.63%of these sequences could mapped to human's reference genome.It is obvious that a large amount of RNA of THP-1 cells is discovered in RNA-seq sequences of intracellular MTB.To avoid the RNA sequences mentioned above from THP-1 cellscontamination,RNA samples of intracellular bacteria were electrophoresis and sample quality could meet the quality characteristics of bacterial RNA.Meanwhile the mRNA distribution had great difference between host mRNA of intracellular MTB and THP-1 cells.These information demonstrates that host mRNA in intracellular bacteria is not from the contamination when separating.According to differences of gene abundance between THP-1 cells and intracellular bacteria,four categories were selected,including:10 mRNA which had high abundance in THP-1 cells,but low in intracellular bacteria;12 mRNA which were high abundance in intracellular bacteria,but low in THP-1 cells;8 mRNA which existed only in in THP-1 cells,but not in intracellular bacteria.The existence of these three types of mRNA reveals that host mRNA in intracellular bacteria is not resulted from contamination as well2.Absolute fluorescence quantitative PCR to verify the THP-1 mRNA invaded into MTB/BCG.To determine the phenomenon of cross-species translocation of mRNA,the mRNA abundance difference between MTB and THP-1 cell were quantitatively compared and verified.We applied absolute fluorescence quantitative PCR to examine the mRNA of MTB and THP-1 cell simultaneously.20 mRNA and 14 miRNA were produced by recombinant method.Fluorescence quantitative results show that mRNA of host translocated into MTB contained both some mRNAs,such as IL1B,IL8 and IER3 etc.which had higher abundance in THP-1 cells than in intracellular bacteria,and other mRNAs,like DHFR,TANK and 7SK etc.,which were less abundant in inTHP-1 cells than in intracellular bacteria.While miRNA of MTB transfered into THP-1 cells,incorporated hsa-miR-142-3P and hsa-miR-30e-5p which were lower abundance in THP-1 cells than in MTB,and hsa-miR-155-5p,hsa-miR-550a-3-5p,chrl-9929(novel miRNA)and chr19-9303(novel miRNA)were lower abundance in MTB than in THP-1 cells.Overall,the phenomenon of host RNA translocation exists from both mRNA and miRNA level.3.Verification of THP-1 cells RNA which did not enter MTB/BCG via reverse transcription PCR.mRNA existed only in THP-1 cells were selected and detected by reverse transcription PCR.The results presents that clear band can be tested in THP-1 cells stimulated by BCG or MTB,but not in intracellular bacteria.This laterally indicates that THP-1 cells RNA enter MTB/BCG have certain selectivity,which is not because of contamination.4.Verification of THP-1 mRNA invaded into MTB/B CG by in situ hybridization test.To directly locate mRNA position,4 high abundance mRNA of THP-1 mRNA invaded into MTB/BCG were choosed and synthesized RNA probes labeled digoxin by transcription in vitro.10ng/?L probe were hybridized with THP-1 cells and intracellular bacteria samples separately to locate the mRNA.Results illustrated that 7SK,DHFR,IL1B and IL8 probe were capable of targeting significant fluorescence signal in both nucleus and cytoplasm of THP-1 cells.Moreover,corresponding fluorescence signals were located in intracellular bacteria samples.Data of in situ hybridization and quantitative PCR consistently provide strong evidence for the phenomenon host RNA of macrophages translocated into intracellular bacteria.