Analysis Of The MRNA-miRNA Expression Profile Of Bovine Monocyte-derived Macrophage Infected With Mycobacterium Avium And Investigation Of Macrophage Apoptosis Regulated By MiRNA-150

Since 1980 s,diseases caused by non-tuberculous mycobacterium(NTM)have increased rapidly,attracting the attention of people to study NTM.Among the NTM,Mycobacterium avium(M.avium)is one of the most common pathogenic bacteria that lead to the diseases of human and animals all over the world.As a result,the precise identification of M.avium is extremely important to evaluate the pathogenicity and epidemiological characteristics.In the four subtypes of M.avium,M.avium subsp.paratuberculosis(MAP)is of the greatest threaten to animal husbandry,which will mainly cause chronic granulomatous enteritis in ruminants,bringing large economic loss for the beef and cow breeding.During the course of MAP infection,macrophages play an important role,in which MAP can survive,proliferate and dissemination.However,the immune response mechanism of macrophages caused by MAP infection is yet unclear.Therefore,the present study takes M.avium as the research object and identifies the subspecie of M.avium strain;further,the expression profile of m RNA and mi RNA of monocyte-derived macrophage purified from bovine,in response to in vitro infection with MAP is sequenced and analyzed;finally,the apoptosis mechanism of macrophages regulated by mi RNA-150 targeting PDCD4 gene is investigated.The primary results are as below:1.Identification of M.avium subspecies and comparative genome analysisThe subspecie of strain HJW and P10 were identified through detection of insertion sequence IS1311,IS900,IS901 and single nucleotide polymorphism of highly-conservative antigen gene 85 B.The results indicated that the strain HJW was M.avium subsp.Silvaticum(MAS)while P10 was MAP.Furthermore,we conducted whole genome sequencing and analysis for the rarely MAS strain HJW and obtained a 0 gap whole genome.The genome involved abundant genetic information,including multiple repetitive sequences,genomic islands and classic VII secretion system of mycobacterium.Combining with the sequencing result,the whole genome sequences of M.avium in the Genebank was comparatively analyzed.The phylogenetic analysis showed that strain HJW was closely related with another Chinese separated strain M.avium RCAD0278,belonging to the same branch.The virulence factor PE/PEE family analysis indicated that except PPE41,PPE42 were specifically belonging to MAP,the other genes distribution showed no obvious specificity.2.Analysis of m RNA expression profile of bovine monocyte-derived macrophage in response to in vitro infection with MAPThe m RNA expression profiles of bovine monocyte-derived macrophage in response to in vitro infection with MAP and that was not infected were analyzed by high-throughput sequencing.A total of 618 genes with significantly different expressed were identified,among which,there were 322 up-regulatory genes and 296 down-regulatory genes.The differentially expressed genes could significantly enrich 145 GO functional groups,including various functional groups relating with immune response of macrophages like cytokine activity,chemotactic factor activity,inflammatory reaction and apoptosis process;in addition,the differentially expressed genes could significantly enrich 54 KEGG signal pathways,including various pathways relating with activating macrophages immune response like NF-?B signal pathway,MAPK signal pathway,TOLL receptor signal pathway,IL-17 signal pathway.Furthermore,the sequencing results were validated by q RT-PCR,which proved the accuracy of the sequencing results.3.Analysis of mi RNA expression profile of bovine monocyte-derived macrophage in response to in vitro infection with MAPThe mi RNA expression profiles of bovine monocyte-derived macrophage in response to in vitro infection with MAP and that was not infected were analyzed by high-throughput sequencing.A total of 68 mi RNAs with differentiated expression were identified,among which,there were 40 mature bovine mi RNA and 21 mi RNA of other animals,and 7 novel mi RNAs;there were 37 up-regulatory mi RNA,31 down-regulatory mi RNA.Combining with m RNA expression profile analysis,the mature bovine mi RNAs target genes were predicted and functional analysis was conducted on the target genes with negative regulation relations.The results showed that the negative regulation target genes could enrich various signal pathways relating with activating the immune response of macrophages,and combining with the mi RNA and m RNA expression profile analysis,the mi RNA-m RNA regulatory network of relative signal pathways was constructed.In addition,the sequencing results were validated by q RT-PCR,which proved the accuracy of the sequencing results.4.mi RNA-150 regulates macrophage apoptosis by targeting PDCD4 gene.First of all,we successfully constructed the overexpression vector of PDCD4 gene and transfect into RAW 264.7 cells.Through flow cytometry,it was validated that PDCD4 gene presented apoptosis-promoting effect on the macrophages.Afterwards,the target relationship between mi R-150 and PDCD4 gene was validated by dual-luciferase reporter test.Furthermore,combining with flow cytometry and western blot,the mi R-150 mimics and inhibitor regulating PDC4 gene regulating macrophages apoptosis effect was validated.At last,the apoptosis detection of si RNA and mi R-150 inhibitor co-transfected cells further showed that mi RNA-150 could regulate the macrophages apoptosis by targeting PDCD4 gene.In conclusion,the present study provides theoretical evidence for explaining the pathogenic mechanism of MAP and the induced immune response of macrophage.Meanwhile,it provides a reference for the study of apoptosis mechanism of macrophages regulated by mi RNA.