羊种布鲁杆菌介导的mmu-miR-149-3p靶基因的初步鉴定

Preliminary identification of the target gene of mmu-miR-149-3p mediated by Brucella melitensis

布鲁杆菌病是由布氏杆菌(Brucella)引起的一种急性或慢性人兽共患传染病,在我国,羊种布鲁杆菌(Brucella melitensis,B.melitensis)最为流行。前期研究发现,mmu-miR-149-3p的潜在靶基因有10个,分别为:Bcl6b、Nos2、Slc7a11、Olr1、Ikbke、Slc31a2、IL1rl1、Dusp16、Ifit1和Rhoc。本试验通过X-treme Gene高效转染试剂,分别将5,50和100 nmol/L,Cy3标记的mimics转染至RAW264.7细胞24 h后,流式细胞仪分析,筛选出最佳转染mimics的浓度;并将mmu-miR-149-3p mimics与mmu-miR-NC mimics,按照最佳转染浓度,转染至RAW264.7细胞24 h后,加入TRIzol裂解细胞,提取总RNA,转录组获得cDNA;利用NCBI primer设计mmu-miR-149-3p的10个潜在靶基因的特异性引物,运用qRT-PCR方法,进行验证试验。结果发现,转染Cy3标记的mimics最佳浓度为100 nmol/L,其阳性率为49.94%,平均荧光强度为18.74;与mmu-miR-NC mimics转染组相比,在mmu-miR-149-3p mimics转染组中,Olr1、Slc7a11和IL1rl1的相对表达量显著降低。结果初步表明,mmu-miR-149-3p的靶基因为Olr1、Slc7a11和IL1rl1。这为揭示mmu-miR-149-3p在B.melitensis侵染巨噬细胞过程中的功能奠定了基础,还为进一步阐释B.melitensis的感染机制以及防控布病提供了参考。

Brucellosis is an acute or chronic zoonotic infectious disease caused by Brucella.Brucella melitensis(B.melitensis),which is the most prevalent strain in China.Previous studies found that there were 10 potential target genes of mmu-miR-149-3 p,namely Bcl6b,Nos 2,Slc7a11,Olr1,Ikbke,Slc31a2,Il1rl1,Dusp16,Ifit1 and Rhoc.In this experiment,5,50,and 100 nmol/L Cy3-labeled mimics were transfected into RAW264.7 cells by X-treme gene high-efficiency transfection reagent.After 24 h,flow cytometry analysis was performed to screen the optimal concentration of transfected mimics.mmu-miRNA-149-3 p mimics and mmu-miRNA-NC mimics were transfected into RAW264.7 cells according to the optimal concentration.24 hours later,the cells were lysised by TRIzol and extracted total RNA,and used to prepare cDNA.NCBI primer was used to design specific primers for 10 potential target genes of mmu-miR-149-3 p,and qRT-PCR was used to verify them.The results showed that the optimum concentration of transfected Cy3-labeled mimics was 100 nmol/L,with a positive rate of 49.94%and an average fluorescence intensity of 18.74.Compared with the mmu-miR-NC mimics transfection group,the relative expression of Olr1,Slc7 a11 and IL1 rl1 in the mmu-mir-149-3 p mimics transfection group decreased significantly.The results showed that the target genes of mmu-miRNA-149-3 p were Olr1,Slc7 a11 and IL1 rl1.This study laid a foundation for revealing the function of mmu-miR-149-3 p in the process of B.melitensis infecting macrophages,and provided a reference for further elucidating the infection mechanism of B.melitensis and preventing and controlling brucellosis.