Analysis of the effects of miRNAs on the expression of cytokines by THP-1 cells using Luminex xMAP technology

Abstract

Objective To investigate the effects of a miRNA family member, let-7e, and a combination of miR-106b and miR-20a on the expression of cytokines by THP-1 cells with Luminex xMAP technology. Methods The efficiency of transfection was evaluated by immunofluorescence assay after transfecting THP-1 cells with micrON™ mimic negative control (Cy3) for 24 h, 36 h and 48 h. The three miRNA mimics (let-7e, miR-106b and miR-20a) were respectively used to transfect the THP-1 cells for 24 h, 36 h and 48 h and the expression of each miRNA was analyzed by qRT-PCR analysis for screening out the optimal transfection time. The transfected THP-1 cells were stimulated with 1 mg/L of LPS for 1 h. The Luminex xMAP technology was used to detect the expression of IL-8, interferon-inducible protein-10 (IP-10), monocyte chemotactic protein 1 (MCP-1), IL-1α, IL-6, IL-10, TNF-α, IFN-α and IFN-β in the supernatants of cell culture. A statistical analysis was performed to analyze the data obtained by using SPSS16.0 software. Results More than 90% of the transfected THP-1 cells were labeled with red fluorescence. The optimal transfection times for let-7e mimic and miR-106b/miR-20a mimics were 48 h and 24 h, respectively. Compared with the corresponding negative control (NC), the expression of IL-8, IP-10 and MCP-1 by THP-1 cells were enhanced after the transfection with let-7e mimic, but were inhibited after the co-transfection with miR-106b and miR-20a mimics. Conclusion The expression of IL-8, IP-10 and MCP-1 were enhanced in let-7e transfected THP-1 cells, but were inhibited in miR-106b and miR-20a co-transfected THP-1 cells. Key words: Luminex xMAP technology; miRNA; Cytokine