miR-146b-5p Plays a Critical Role in the Regulation of Autophagy in ∆per Brucella melitensis-Infected RAW264.7 Cells

Abstract EN

Brucella-caused brucellosis is one of the most widespread worldwide zoonoses.

Lipopolysaccharide (LPS) of Brucella, which functions as pathogen-associated molecular patterns (PAMPs), is an important virulence factor that elicits protective antibodies.

Per of B.

melitensis 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 Brucella.

In this study, we observed that autophagy was inhibited in RAW264.7 cells infected with Brucella melitensis ∆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 B.

melitensis M5-90 ∆per.

The results suggested that mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-146b-5p, and mmu-miR-3473a were upregulated and mmu-miR-30c-5p was downregulated.

During B.

melitensis M5-90 ∆per infection, the increased expression of miR-146b-5p inhibited the autophagy activation in RAW264.7 cells.

Using a bioinformatics approach, Tbc1d14 was predicted to be a potential target of miR-146b-5p.

The results of a luciferase reporter assay indicated that miR-146b-5p directly targeted the 3′-UTR of Tbc1d14, and the interaction between miR-146b-5p and the 3′-UTR of Tbc1d14 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, IFNγ-inducible p47 GTPase 1 (IIGP1), nuclear receptor binding protein 2 (Nrbp2), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1), and immunity-related GTPase family M member 1 (Irgm1), were obtained.

Our findings provide important insights into the functional mechanism of LPS of B.

melitensis.