Inducible MiR-203 Feedback Negatively Regulates TNF-α And IL-6 Production In Heat Killed Candida Albicans Stimulated Monocyte Derived Dendritic Cell Via Targeting TLR4

Part 1 MicroRNA profile alternation of monocyte derived dendritic cells after heat killed Candida albicans stimulationMicroRNA array was used to determine the microRNA profile alternation of monocyte derived dendritic cells stimulated with heat killed Candida albicans. As a result, 62 microRNAs was up-regulated, while 4 microRNAs was down-regulated after heat killed Candida albicans stimulation. Bioinformational approaches, including Gene Ontology, pathway, regulation net and transcription factors analysis, were used to analyze the significance of the differentiated expressed microRNA. MiR-203, one of the most significantly differentiated expressed microRNA, was selected as the target microRNA for the further research.Part 2 Heat killed Candida albicans induced IL-6 and TNF-αproduction from monocyte derived dendritic cells was negatively regulated by mir-203By quantitative RT-PCR, we found the relative expression of miR-203 in monocyte derived dendritic cells was elevated after stimulated with heat killed Candida albicans. The inducible expression of miR-203 was Dectin-1, but not TLR4 dependent. Heat killed Candida albicans stimulation also caused IL-6 and TNF-αproduction through Dectin-1 and TLR4. More importantly, by gain-and loss-of-function, miR-203 was found as a negative regulator of IL-6 and TNF-αproduction caused by heat killed Candida albicans. Therefore, we demonstrate that inducible miR-203 feedback negatively regulates IL-6 and TNF-αproduction in monocyte derived dendritic cells treated with heat killed Candida albicans.Part 3 TLR4 is a target of miR-203 in monocyte derived dendritic cellsTo clarify the molecular mechanisms of the IL-6 and TNF-αproduction regulated by miR-203, bioinformational approaches were used to predict the possible target of miR-203. TLR4, one of the well characterized patter recognization receptor for Candida albicans immune sense, was predicted as the target of miR-203. As showed by dual-luciferase reporter assay, miR-203 could interact with the 3'UTR of TLR4 mRNA. In addition, heat killed Candida albicans stimulation caused the down expression of TLR4, this can be enhanced by miR-203 mimics transfection, while partially blocked by miR-203 inhibitor transfection in monocyte derived dendritic cells. Therefore, we demonstrate TLR4 is target of miR-203 in monocyte derived dendritic cells during heat killed Candida albicans stimulation.Part 4 Knocking down TLR4 expression in monocyte derived dendritic cells impaired heat killed Candida albicans induced IL-6 and TNF-αproduction, but has no effect on miR-203 expressionTo determine whether miR-203's negative effect on IL-6 and TNF-αproduction triggered by heat killed Candida albicans was specifically mediated by targeting TLR4, siRNA approach was used to silence the expression of TLR4 in dendritic cells. Similar to the effect of miR-203, TLR4 siRNA transfection partially inhibit IL-6 and TNF-αproduction induced by heat killed Candida albicans, but has no effect on miR-203 expression. Therefore, we demonstrate miR-203 negatively regulate IL-6 and TNF-αwas mediated by targeting TLR4.