miR‐146b‐5p Plays a Critical Role in the Regulation of Autophagy in ∆per <i>Brucella melitensis</i>‐Infected RAW264.7 Cells

Abstract

<i>Brucella</i>-caused brucellosis is one of the most widespread worldwide zoonoses. Lipopolysaccharide (LPS) of <i>Brucella</i>, which functions as pathogen-associated molecular patterns (PAMPs), is an important virulence factor that elicits protective antibodies. <i>Per</i> of <i>B</i>. <i>melitensis</i> is involved in the biosynthesis of the O-side chain of LPS. Autophagy is a crucial element of the innate immune response against intracellular pathogens including <i>Brucella</i>. In this study, we observed that autophagy was inhibited in RAW264.7 cells infected with <i>Brucella melitensis</i> ∆per. And, a high-throughput array-based screen and qRT-PCR validation were performed to identify the differentially expressed miRNAs in RAW264.7 cells infected with <i>B. melitensis</i> M5-90 ∆per. The results suggested that <i>mmu-miR-146a-5p</i>, <i>mmu-miR-155-5p</i>, <i>mmu-miR-146b-5p</i>, and <i>mmu-miR-3473a</i> were upregulated and <i>mmu-miR-30c-5p</i> was downregulated. During <i>B. melitensis</i> M5-90 ∆per infection, the increased expression of <i>miR-146b-5p</i> inhibited the autophagy activation in RAW264.7 cells. Using a bioinformatics approach, <i>Tbc1d14</i> was predicted to be a potential target of <i>miR-146b-5p</i>. The results of a luciferase reporter assay indicated that <i>miR-146b-5p</i> directly targeted the 3'-UTR of <i>Tbc1d14</i>, and the interaction between <i>miR-146b-5p</i> and the 3'-UTR of <i>Tbc1d14</i> was sequence-specific. High-throughput RNA-Seq-based screening was performed to identify differentially expressed genes in Tbc1d14-expressing RAW264.7 cells, and these were validated by qRT-PCR. Among the differentially expressed genes, four autophagy associated genes, <i>IFNγ-inducible p47 GTPase</i> 1 (<i>IIGP1</i>), <i>nuclear receptor binding protein 2</i> (<i>Nrbp2</i>), <i>transformation related protein 53 inducible nuclear protein 1</i> (<i>Trp53inp1</i>), and <i>immunity-related GTPase family M member 1</i> (<i>Irgm1</i>), were obtained. Our findings provide important insights into the functional mechanism of LPS of <i>B. melitensis</i>.