Molecular Mechanism Of Brucella Per Gene Affecting Cell Autophagy

Zoonosis Brucellosis is mainly caused by brucella,LPS(Lipopolysaccharide)is a major virulence factor of brucella.Per gene encodes the synthesis of the perosamine synthetase,which involved in synthsis of smooth brucella lipopolysaccharide O side chain.when per gene deleted,it can not synthesize LPS and brucella virulence was attenuated or disappeared,and resulting in the smooth type brucella changed into rough.MicroRNAs(miRNAs)is a class of non-coding single-stranded small RNA.The length of the miRNAs is about 18-24 nucleotides.miRNAs combined the seed sequence of the target gene 3'UTR(3' untranslated regions,3'UTR),which mainly involved in transcriptional regulation.Autophagy is a highly conserved process in eukaryotes,the double membrane cystic structure wrapped the cytoplasm,damaged organelles,cell invasion of pathogenic microorganisms,and which was sent to the lysosome or vacuole,it was enzymatic hydrolysis into macromolecules and impurities.macromolecules were recovered and reused by cells,while the impurities were discharged to the extracellular.In this study,we used B.melitensis M5-90 and M5-90-?per deletion strains to infect RAW264.7 cells,and found that miR-146b-5p was significantly upregulated,it significantly reduced the level of autophagy at the same time,revealed in B.melitensi.s M5-90 infection process,miR-146b-5p target binding Tbcld14 to mediate the molecule mechanism of RAW264.7 autophagy.Methods1)Using homologous recombination to construct M5-90-?per deletion strain,B.melitensis M5-90 and M5-90-Aper deletion infected RAW264.7 for 4 h,the miRNA expression profiles identified by miRNA microarray-single,the mRNA expression profiles identified by agilent microarray,and then using miRNA-mRNA expression joint analysis,qRT-PCR validated the miRNA and it's target genes,transfection mimic and dual luciferase reporter gene experiments were further used to validate the joint analysis.2)B.melitensis M5-90 and M5-90-?er deletion infected RAW264.7 for 4 h,western blot to detect LC3-?/? protein and transmission electron microscopy to detect the number of autolysosomes were performed to analysis autophagy.3)RAW264.7 cells were seeded 5 × 105 cells/well to 12-well plate at 0 h,trasfected with miR-NC mimic,miR-146b-5p mimic,miR-NC inhibitor and miR-146b-5p inhibitor at 24 h,infected by B.melitensis M5-90 at 44 h,and harvested at 48 h.Western blot to detect LC3-?/? protein and transmission electron microscopy to detect the number of autolysosomes were performed to analysis autophagy.4)Packaging the over expression adenovirus of Tbc1d14 and GFP.RAW264.7 cells were seeded 5 × 105 cells/well to 12-well plate at 0 h,infected by Ad-Tbc1d14 and Ad-GFP adenovirus at 12 h,trasfected with miR-NC mimic,miR-146b-5p mimic,miR-NC inhibitor and miR-146b-5p inhibitor at 24 h,infected by B.melitensis M5-90 at 44 h,and harvested at 48 h.Western blot to detect LC3-II/I protein and transmission electron microscopy to detect the number of autolysosomes were performed to analysis autophagy.5)RAW264.7 cells were infected by Ad-Tbcldl4 and Ad-GFP adenovirus for 36 h,performed DGE sequencing,GO was used for functional enrichment analysis to find autophagy pathway related and significantly differentially expressed genes.Results1)PCR amplified the left and right homologous of per gene by the template of B.melitensis M5-90 genome,kana gene replaced per gene CDS,successfully constructed the homologous recombination plamid of pGEM-?per,electric tranfection with B.melitensis M5-90 competent cells,PCR and sequencing was used to indentify the correct clones,the positive clone named M5-90-?per.2)Probe methods for qRT-PCR validatated the miRNA expression profile chip,it was found that miR-146a-5p,miR-155-5p,miR-146b-5p,miR-3473a and miR-30c-5p were corrected with microarray results.Agilent microarray results showed,one or more fold-change genes were 869,using miRNA-mRNA expression conjoint analysis,the results obtained 18 pairs of miRNA-target genes relationship,qRT-PCR validation of target genes,transfection mimic experiment and dual luciferase reporter gene assay,and eventually found that miR-146b-5p and Tbcldl4 was the correct miRNA-target gene relationship.3)B.melitensis M5-90 and M5-90-?per deletion infected RAW264.7 for 4 h,western blot to detect LC3-?/? protein and transmission electron microscopy to detect the number of autolysosomes were performed to analysis autophagy.The results showed that M5-90-?per infection group RAW264.7 auophagy level was significantly reduced.4)Overexpression of miR-146b-5p significantly reduced the level of B.melitensis M5-90 midiated RAW264.7 autophagy.Suppression endogenous of miR-146b-5p can significantly increased the level of B.melitensis M5-90 midiated RAW264.7 autophagy.5)Under conditions of B.melitensis M5-90 infection,overexpression Tbc1d14 resulted in the miR-146b-5p mediating RAW264.7 autophagy.6)DGE sequenced RAW264.7 after overexpression Tbcldl4,there are 612 significantly differentially up-regulated genes,and 175 significantly differentially down-regulated genes.By GO functional enrichment analysis,we preliminary found that there are 4 genes associated with autophagy pathway,it's Iigp1,Nrbp2,Trp53inp1 and Irgml.Among them,Iigpl,Trp53inpl and Irgml upregulated,and Nrbp2 downrugulated.ConclusionsThis study successfully constructed B.melitensis M5-90 strain per gene deletion strain.B.melitensis M5-90 and M5-90-?per deletion strain infected RAW264.7 for 4 h,respectively performed miRNA microarray-single chip and agilent microarray gene expression profiling chip,and used miRNA-mRNA expression conjoint analysis,found that miR-146b-5p upregulated and modulated the expression of target gene Tbcld14.B.melitensis M5-90 and M5-90-?per deletion strain infected RAW264.7 for 4 h,we also found the level of RAW264.7 autophagy in M5-90-?per deletion strain infected group significantly decreased,under the condition of B.melitensis M5-90 infection.We analyzed the RAW264.7 autophagy after inhibition of endogenous and-overexpression of miR-146b-5p,respectively.The results showed that miR-146b-5p directly negative correlation regulate RAW264.7 autophagy.Packaging overexpressed Tbc1d14 adenovirus,under the condition of B.melitensis M5-90 infection,we found that overexpression Tbc1d14 can result in increasing of the RAW264.7 autophagy level mediated by miR-146b-5p.After overexpressing Tbc1d14 in RAW264.7,DGE sequenceing analysis the differentially expressed genes.The datas showed,there are 612 significantly differentially up-regulated genes,and 175 significantly differentially down-regulated genes.GO functional enrichment analysis the DGE sequence data,we preliminary found that there are 4 genes associated with autophagy pathway,it's Iigpl,Nrbp2,Trp53inpl and Irgml.Among them,Iigpl,Trp53inpl and Irgml upregulated,and Nrbp2 downrugulated.The data showed that miR-146b-5p target Tbc1d14 and negative correlation mediated RAW264.7 autophagy under the condition of B.melitensis M5-90 infection.And we preliminary found that Tbcldl4 may regulate the Iigpl,Nrbp2,Trp53inpl and Irgml downstream genes to mediate RAW264.7 autophagy.