Fengyang Wang

Downregulation of miR-27a* and miR-532-5p and Upregulation of miR-146a and miR-155 in LPS-induced RAW264.7 Macrophage Cells

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the epigenetic regulation of cellular processes. To identify more miRNAs which are involved in the macrophage inflammatory response to lipopolysaccharide (LPS) stimulation and dissect the mechanisms more clearly, microRNA profiling of LPS-treated RAW264.7 macrophage cells 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 untreated control, two microRNAs (miR-146a and miR-155) with more than twofold higher expression and two microRNAs (miR-27a* and miR-532-5p) with twofold lower 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 upregulation and downregulation of miRNA to the differential expression of inflammation-related candidate target gene during LPS-induced inflammation. Our findings may provide the basic information for the precise roles of miRNAs in the macrophage inflammatory response to LPS stimulation in the future.

siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells.

Innate immunity plays a key role in protecting a host against invading microorganism, including Gram-negative bacteria. Cluster of differentiation antigen 14 (CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor 4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14 as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7 cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14 gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14 expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule (mCD14–siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically, was identified, RAW264.7 cells pretreated with mCD14–siRNA-224 were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) and the NO production were evaluated. The results indicated that mCD14–siRNA-224 effectively inhibited TNF-α, MIP-2, and IL-6 release and NO production from LPS-stimulated RAW 264.7 cells by down-regulating mRNA accumulation and protein expression of mCD14 specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.

Inhibition of mCD14 inhibits TNFα secretion and NO production in RAW264.7 cells stimulated by Brucella melitensis infection.

Brucellosis, caused by Brucella spp., 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, CD14, 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. The aim of this study was to explore the possibility of using RNA interference (RNAi) targeting mCD14 as a strategy for inhibiting the secretion of tumor necrosis factor alpha (TNFα) and the production of nitric oxide (NO) from Brucella-stimulated RAW264.7 cells and attenuating damaging immune activation. The current study stably incorporated mCD14-shRNA-224 into the RAW264.7 cell line by lentiviral gene transfer to successfully knockdown mCD14, and was then challenged with Brucella melitensis M5-90. The secretion of TNFα, interleukin (IL)-12, CXCL1/KC, and inducible nitric oxide synthase (iNOS) protein expression, and NO production were evaluated. The mCD14-shRNA-224 knockdown was shown to effectively inhibit B. melitensis M5-90-stimulated TNFα release, iNOS protein expression, and NO production, but no significant differences were observed for IL-12 and CXCL1/KC. These findings provide the basis for the development of RNAi-based prophylaxis and therapy for B. melitensis induced inflammatory disease.

Identification of MicroRNAs Dysregulated in CD14 Gene Silencing RAW264.7 Macrophage Cells

A cluster of differentiation antigen 14 (CD14) is involved in lipopolysaccharide (LPS)-induced proinflammatory cytokine release and LPS-induced septic shock. MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the epigenetic regulation of cellular process and bacterial infection. Our previous study indicated that siRNA against CD14 effectively inhibited LPS-induced tumor necrosis factor alpha, chemokine (C-X-C motif) ligand 2, interleukin-6 release, and NO production. To identify miRNAs which are affected by CD14 gene silencing and dissect the mechanisms of the attenuating of LPS-induced damaging immune activation more clearly, based on the CD14 knockdown RAW264.7 macrophage cell line established in our previous study, miRNAs expression profiling of CD14 knockdown RAW264.7 cells were analyzed with miRNA microarray and validated by qRT-PCR, the potential targets were predicted and subjected to gene ontology (GO) pathway and biological processes analysis. We demonstrated for the first time that CD14 knockdown significantly changed the expression of 199a-3p, miR-199a-5p, and miR-21-5p in RAW264.7 cells, and significantly enriched GO terms in the predicted target genes of these miRNAs were apoptosis process, immune response, inflammatory response, innate immune response, anti-apoptosis, cytokine production, and cytokine-mediated signaling pathway. These findings may improve our understanding about functional mechanism of miRNAs in the attenuating of LPS-induced damaging immune activation more clearly.

Identification of miR-221 and -222 as important regulators in genotype IV swine hepatitis E virus ORF3-expressing HEK 293 cells

Hepatitis E virus (HEV) has emerged as an important cause of epidemic and sporadic acute viral hepatitis worldwide, which is a major public health challenge. A better understanding of the interaction between the virus and the host cell would be very helpful for its therapy. Swine HEV (SHEV) open reading frame 3 (ORF3) is a regulatory protein that alters the activity of selected transcription factors and cytoplasmic signaling pathways. MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein-coding genes and represent an interesting lead to study SHEV infection and to identify new therapeutic targets. To explore how SHEV ORF3 affects miRNAs in host cells, we used miRNA array analysis to compare the expression patterns of miRNAs in stable cell lines that expressed or did not express SHEV ORF3. We found a significant down-regulation of miR-221 and -222 in ORF3 expressing human embryonic kidney 293 cell line. Among the 116 candidate targets genes of miR-221 and -222 that we detected in silico, we demonstrated that the expression of the cyclin-dependent kinase inhibitor 1B, also named p27kip1, was directly regulated by these miRNAs. We hypothesize that SHEV ORF3-induced miR-221/222 downregulation enhances p27kip1 expression in HEK293 cells. This provides new avenues for future exploration of the precise roles of miRNAs in SHEV infection.

Effective inhibition of mRNA accumulation and protein expression of H5N1 avian influenza virus NS1 gene in vitro by small interfering RNAs

Avian influenza has emerged as a devastating disease and may cross species barrier and adapt to a new host, causing enormous economic loss and great public health threats, and non-structural protein 1 (NS1) is a multifunctional non-structural protein of avian influenza virus (AIV) that counters cellular antiviral activities and is a virulence factor. RNA interference (RNAi) provides a powerful promising approach to inhibit viral infection specifically. To explore the possibility of using RNAi as a strategy against AIV infection, after the fusion protein expression plasmids pNS1-enhanced green fluorescent protein (EGFP), which contain the EGFP reporter gene and AIV NS1 as silencing target, were constructed and NS1-EGFP fusion protein expressing HEK293 cell lines were established, four small interfering RNAs (siRNAs) targeting NS1 gene were designed, synthesized, and used to transfect the stable cell lines. Flow cytometry, real-time quantitative polymerase chain reaction, and Western blot were performed to assess the expression level of NS1. The results suggested that sequence-dependent specific siRNAs effectively inhibited mRNA accumulation and protein expression of AIV NS1 in vitro. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for AIV infection.

Up-regulation of TDAG51 is a dependent factor of LPS-induced RAW264.7 macrophages proliferation and cell cycle progression

Abstract Context: As a component of the outer membrane in Gram-negative bacteria, lipopolysaccharide (LPS)-induced proliferation and cell cycle progression of monocytes/macrophages. It has been suggested that the proapoptotic T-cell death-associated gene 51 (TDAG51) might be associated with cell proliferation and cell cycle progression; however, its role in the interaction between LPS and macrophages remains unclear. Objective: We attempted to elucidate the role(s) of TDAG51 played in the interaction between LPS and macrophages. Materials and methods: We investigated TDAG51 expression in RAW264.7 cells stimulated with LPS and examined the effects of RNA interference-mediated TDAG51 down-regulation. We used CCK-8 assay and flow cytometry analysis to evaluate the interaction between TDAG51 and LPS-induced proliferation and cell cycle progression in RAW264.7 cells. Results: Our findings indicate that TDAG51 is up-regulated in LPS-stimulated RAW264.7 cells, the TDAG51 siRNA effectively reduced TDAG51 protein up-regulation following LPS stimulation in RAW264.7 cells, the significant changes of the proliferation and cell cycle progression of RAW264.7 cells in TDAG51 Knockdown RAW264.7 cells treated with LPS were observed. Conclusion: These findings suggested that TDAG51 up-regulation is a dependent event during LPS-mediated proliferation and cell cycle progression, and which increase our understanding of the interaction mechanism between LPS and macrophages.