The Influence Of Host MiRNA-363-5p On The Pathogenicity Of Brucella

Brucellosis is an important Zoonosis caused by Brucella,which brings great threat to the development of livestock and the health of human and animals.It has developped multiple strategies to evade host immune response,establishing persistent infection in host.MicroRNAs(miRNAs)are small non-coding endogenous RNAs and regulate gene expression at post-transcription level and play an important regulatory role in the host.Previously,we have obtained that miRNAs' expression profile from spleen of BALB/c mice infected with B.melitensis virulent(M28)and vaccine(M5-90)strain.And we found that the targets of differentially expressed microRNAs regulated a wide range of biological processes including apoptosis,autophagy and inflammatory response.Preliminary study found that microRNA-363-5p participated in macrophage apoptosis induced by Brucella.To explore the role of differentially expressed microRNAs in macrophages infected by Brucella and screen differentially expressed microRNAs involved in Brucella replication in vitro and apoptosis of macrophage induced by Brucella.Macrophage(RAW264.7)transfected by microRNA mimics and inhibitors,after infection of M28 and M5-90,bacteria number in RAW264.7 was counted and cell state was observed.The results showed that overexpression of miRNA-29c-5p or inhibition of miRNA-335-5p and miRNA-329-3p significantly reduced the ability of intracellular replication of Brucella in RAW264.7(p<0.05).When overexpressing miRNA-676-3p,miRNA-674-5p and miRNA-18b-5p,after infection of M28,the cells grew slowly.We hypothesized that miRNA-676-3p,miRNA-674-5p and miRNA-18b-5p could participate in macrophage apoptosis,and the mechanism of it needed to be explored.The targets of microRNA-363-5p were tested and verified using dual luciferase reporter system and Western blot based on the previous work.The results of dual luciferase reporter gene system showed that the expression of firefly luciferase was significantly reduced by co-transfection of plasmid pmirGLO-Prkacb,pmirGLO-Lgf1,pmirGLO-Pdpk1,pmirGLO-Pik3 cg,pmirGLO-Cycs,pmirGLO-Wnt7 a and pmirGLO-Sema6 a and microRNA-363-5p mimic(p<0.001).When the seed sequence of targets mutated,the expression of firefly luciferase can be recovery.Based on the results of dual luciferase gene reporter system,further study verified that the expression of Prkacb,Cycs and Sema6 a in macrophages were significantly decreased after transfection of microRNA-363-5p by Western blot.To summarize,the results showed that Prkacb,Cycs and Sema6 a were the targets of microRNA-363-5p.In order to explore the effect of microRNA-363-5p on B.melitensis replication in the host,BALB/ C mice were used as an animal model by overexpression or inhibition of the expression of miR-363-5p.After infecting mice,bacteria isolated in the spleen was counted.Over expression or inhibition of the expression of miR-363-5p in mice,spleen weight of mice did not change.And there were no difference between the number of bacteria isolated in the spleen of control mice and mice by overexpression of miRNA-363-5p.However,inhibiting expression of miRNA-363-5p in mice,bacteria number was significantly lower than control mice(p<0.01).The results showed that inhibition of the expression of miRNA-363-5p can significantly promote the replication of Brucella in mice.In addition,to explore the effect of OmpR(One of EnvZ/OmpR two-component regulatory system)on the growth and replication of Brucella.An B.melitensis deletion strain was constructed by replacing OmpR gene in the M28 with kanamycin resistance gene based on homologous recombination,named M28?OmpR.Growing Test showed that velocity of M28?OmpR was significantly lower than M28 on solid medium and M28?OmpR-com could recover growth(p<0.001).The intracellular survival rate of M28?OmpR in macrophage was one hundred times lower than M28 and M28?OmpR-com(p<0.001).The results of mouse virulence survival were shown that the advantage of M28?OmpR was smaller than M28,bacteria isolated in the spleen was eight times lower than that in the mice infected with the parental bacteria at 4-week post infection(p<0.001).Furthermore,spleen weight of mice infected with M28?OmpR was significantly lower than that in the mice infected with M28(p<0.001).In conclusion,Deletion of OmpR gene inhibited the growth of B.melitensis M28 and significantly reduced its capacity to replicate in macrophages and in mice.In summary,the study demonstrated that overexpression of miRNA-29c-5p or inhibition of miRNA-335-5p and miRNA-329-3p significantly reduced the ability of intracellular replication of Brucella in RAW264.7.MiRNA-363-5p participated in apoptosis of macrophage by regulating Prkacb,Cycs and Sema6 a.Inhibition of the expression of miRNA-363-5p could significantly promote the replication of Brucella in mice.Deletion of OmpR gene inhibited the growth of B.melitensis M28 and significantly reduced its capacity to replicate in macrophages and in mice.This study provided a new idea for curing brucellosis by gene therapy and laid the foundation for the further study of interaction between Brucella and host.