MicoRNA Expression Analysis Of MCD14 Knockdown RAW264.7 Cells Infected By Brucella Melitensis

Brucellosis, caused by Brucella, is an important disease affecting not only human health, but also a number of animal species around the world. A receptor for LPS of Brucella and important innate immune molecule, CD 14, has been implicated in the initiation of the inflammatory response to sepsis. Evidence indicates that upstream inhibition of the LPS initiated inflammatory pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) technology is a approach to reduce target gene expression efficient and specificly, The aim of this study was to explore the possibility of using RNA interference (RNAi) targeting mCD14 as a strategy for studying the changes of the microRNAs from Brucella-stimulated RAW264.7 cells.The current study stably incorporated mCD14-shRNA-224 into the RAW264.7 cell line by lentiviral gene transfer to successfully knockdown mCD14, and the cells named 224.3.We then tested the inhibition effects of mCD14 expression though RNAi by real-time quantitative RT-PCR and Western blot. Results show that RNAi is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically. To identify more miRNAs which are involved in the macrophage inflammatory response to Brucella stimulation and dissect the mechanisms more clearly, microRNA profiling of Brucella-treated 224.3 was performed by initial high-throughput array-based screen and further real-time RT-PCR validation; bioinformatics approaches were used to analyze the target genes of the differentially expressed miRNAs.Compared to the RAW264.7 cell and negative control, two microRNAs (mmu-miR-199a-3p and mmu-miR-183-5p) higher expression were detected by array-based screen, which can be validated by qRT-PCR, and more than 1,000 candidate target genes were detected by at least of one of four different algorithms (TargetScan, PicTar, miRDB, and microRNA.org); with Gene Ontology classification, we were able to correlate the up-regulation miRNA to the differential expression of inflammation-related candidate target gene during Brucella-induced inflammation. Our findings may provide the basic information for the precise roles of miRNAs in the macrophage inflammatory response to Brucella.