Screening And Verification Of Circulating MiRNA Related To Brucella Suis Infection

Brucellosis is a severe zoonotic disease caused by Brucella,It is widespread in more than 160 countries and regions all over the world,and poses a great threat to the development of animal husbandry and human health worldwide.At present,the diagnosis methods of brucellosis can be divided into etiological diagnosis and serological diagnosis,However,the two methods have some disadvantages: the pathogen diagnosis has some problems,such as the difficulty of separation and operation,the low success rate,the long time of pathogen culture and the bio-safety risk in the process of separating pathogen.The serological diagnosis has some problems such as easy cross-reaction and high cost between the agglutinating antigen and many kinds of antisera(E.coli O157,Yersinia O9,etc.)which have similar antigen epitopes to Brucella.MicroRNA(miRNA for short)is a kind of endogenous non-coding small molecule RNA,which exists widely in nematodes,fruit flies,plants and eukaryotes,including human beings,and has a long history of about 22 nm.With further research,it has been found that miRNA can be released from the cell and is widely and steadily present in extracellular fluids,including serum,plasma,saliva,urine,and even milk,This type of miRNA is collectively referred to as circular miRNA(circulating microRNA).Circulating miRNA has many advantages,such as stable existence,conserved evolution among species and various detection methods.As a new diagnostic marker of diseases,cyclic miRNA has been studied in many fields of human medicine.Therefore,based on the defects of the two diagnostic methods for brucellosis and the current status of circulating miRNA in the field of diseases,We hope to explore the relative changes of miRNA expression in some circulating miRNA after Brucella infection in the host,and to explore the possibility of using such circulating miRNA as a diagnostic marker of Brucella infection.In this study,a BALB/c mouse model of Brucella infection was established,BALB/ c mice were infected with S2 strain and S1330 strain of Brucella suis,and the same number of blank control group were set up.On the 7th,14 th,21st and 28 th day after infection,5 mice in each group were collected from each group.Blood samples were collected from the eyeballs of each mice,The change trend of serum antibody level,spleen weight and spleen bacterial count in three groups of mice at four time points were obtained by the weight of spleen,the weight of spleen and the number of spleen bacteria in the spleen after aseptic enucleation of the spleen and the weight of sterilized ground spleen,Based on the trends reflected by the three data sets,the serum samples from day 28 were determined for transcriptional sequencing.S1330 infection group and blank control group showed significant changes in the expression of 15 miRNA in serum,of which 9 were up-regulated and 6 were down-regulated.The expression of miRNA in serum of S2 strain infection group and blank control group had significant changes,of which 4 were up-regulated and 7 were down-regulated.The expression of miRNA in serum of S1330 infection group and S2 infection group was significantly changed,one of them was up-regulated and two down-regulated.According to the results of transcriptional sequencing,we selected 10,2 and 2 miRNA from the three control groups,which showed significant changes in expression.In the miRBase database,the sequence information was queried and the primers were designed to detect the miRNA.Experiments were carried out by miRNA extraction,reverse transcription,fluorescence quantitative PCR and extended sample detection in mouse serum,S1330 infection and blank control groups were selected to match the sequencing results of the transcript group and the expression of miRNA was significantly different(up-regulated)in the S1330 strain.mmu-miR-10b-5p.Meanwhile,we also studied the expression of mmu-miR-10b-5p in RAW264.7-infected porcine Brucellosis S1330 strain in mouse macrophages,The results showed that the expression of mmu-miR-10b-5p in macrophages was significantly different after infection with S1330 strain for 48 hours in control and infection groups.Afterwards,we predicted the downstream target of mmu-miR-10b-5p in macrophages.In the predicted 52 downstream target genes,we verified that the relative expression levels of the 6 genes were significantly down-regulated.The functions of these 6 genes are mostly related to apoptosis and inflammatory responses.Finally,features based on the highly conserved miRNA sequence,We detected the expression of bta-miR-10b-5p in positive and negative bovine serum samples.Unfortunately,the expression of bta-miR-10b-5p did not show significant difference.In summary,through the study of this paper,a miRNA with a significant difference(up-regulation)in the expression of S1330 infection group and blank control group was successfully screened,which provide ideas for the study of circulating miRNAs as a diagnostic marker for Brucella infection.