The Regulatory Effect Of MiR-142-3p On Inflammatory Factors Released By THP-1Cells

Background:Sepsis is the systemic inflammatory response syndrome caused by bacterial, viral andparasitic infections. If proper treatments are not provided promptly, patients are likely tosuffer from septic shock and multiple organ dysfunction syndrome (MODS) with poorprognosis and high mortality rate at30%-50%. The invasion of microbe initiates a varietyof inflammatory factors release. Sepsis is characterized by the immune dysfunctionincluding amplification of inflammatory cascade and overwhelming release ofinflammatory factors, which eventually lead to cell injury, apoptosis and organdysfunction. However, there are still no specific treatments for sepsis and the cost fortreatments is very expensive. In the past20years, many researches on sepsis therapieshave failed due to unsatisfactory results; for instant, monoclonal antibodies towards tumornecrosis factor-α (TNF-alpha), interleukin-1(IL-1) and other cytokines. In order to controlinflammatory response earlier, researchers are now looking for the upstream regulatoryfactors of inflammation. MicroRNA (miRNA) is a short ribonucleic acid (RNA) moleculewith very few nucleotides (an average of21-25). MiRNAs are post-transcriptionalregulators that bind to complementary sequences on target messenger RNA (mRNA)transcripts, usually resulting in translational repression or target degradation and genesilencing. In recent years, more and more studies found that miRNA involved in theregulation of sepsis. It inhibits proinflammatory gene expression and regulates theproliferation, differentiation and apoptosis of inflammatory cells. Among the miRNAs,miR-142-3p was demonstrated that it varies in quantity significantly in sepsis model and might be a regulator in sepsis. But the exact mechanism of miR-142-3p in sepsis is stillunclear.Objective:1. Our study aims to investigate the role of miR-142-3p in inflammatory factorsrelease by THP-1cells.2. Our study would provide experimental basis for discovering new pathway toregulate the inflammatory factors release by immune cells.Method:1. THP-1cells was cultured and dyed with Propidium Iodide to test survival rate.2. THP-1cells were stimulated for24hours with0.5μg/ml and2.0μg/mllipopolysaccharides (LPS). The concentrations of TNF-α, interleukin-6(IL-6) andmonocyte chemotactic protein-1(MCP-1) in the cultured supernatant weredetected by enzyme-linked immunosorbent assay (ELISA).3. THP-1cells were transfected with the fluorescence RNA. The transfectionefficiency of lipofectamine IMAX reagents was observed by fluorescencemicroscopy. The expressive levels of miR-142-3p in THP-1cell aftertransfection with miR-142-3p mimic or inhibitor (100nM) were detected byquantitative real time polymerase chain reaction (qRT-PCR).4. THP-1cells were transfected with miRNA-142-3p mimics and cultured for48h.The concentrations of TNF-α, IL-6and MCP-1in the cultured supernatant weredetected by ELISA.5.(1).THP-1cells were transfected with miRNA-142-3p repressor (inhibitor) andcultured for48h.(2).After transfection, THP-1cells were stimulated by0.5μg/ml and2.0μg/ml LPSfor24h. The concentrations of TNF-α, IL-6and MCP-1in the cultured supernatant were detected by ELISA.Result:1. It showed that the concentrations of TNF-α, IL-6and MCP-1with0.5μg/ml or2.0μg/ml LPS stimulated all increase significantly when comparedwith the control group（P＜0.01, respectively）.Meanwhile, the secretion of thecytokines above presented a dose-dependent response to LPS stimulated. Theconcentrations of TNF-α, IL-6and MCP-1in2.0μg/ml LPS group are significantlyhigher than in0.5μg/ml LPS group（TNF-α：P＜0.01; IL-6、MCP-1：P＜0.05,respectively）.2. The expressive level of miR-142-3p elevated significantly in THP-1cells with100nM miR-142-3p mimic transfected when compared with negative control (NC)group(P<0.05). The expressive level of miR-142-3p reduced after miR-142-3pinhibitor transfection, but there are no significant difference between transfectedgroup and untransfected group (P>0.05).3. After100nM miR-142-3p mimic transfection, ELISA result showed that TNF-α, IL-6and MCP-1levels in cultured supernatant elevated in different levels,with statistically difference (vs. NC group,TNF-α: P<0.05; IL-6, MCP-1: P<0.01,respectively).4. With100nM miR-142-3p inhibitor transfected,the concentrations of TNF-α, IL-6andMCP-1showed no statistical difference between groups（P＞0.05, respectively）.5. The concentration of IL-6was significantly reduced in cultured supernatant of THP-1cells which were pre-transfected with miR-142-3p inhibitor and stimulated with0.5μg/ml LPS compared to NC group (P<0.05), but not with2.0μg/ml LPS (P>0.05).The concentrations of TNF-α and MCP-1in THP-1cells cultured supernatant withmiRNA-142-3p inhibitor pre-transfected and LPS-stimulated also have a slight decrease, but it does not show significant statistical difference（P＞0.05,respectively）.Conclusion:1. LPS stimulates THP-1cells to release TNF-α、IL-6and MCP-1.2. Overexpression of miR-142-3p enhances the release of TNF-α, IL-6and MCP-1inTHP-1cells.3. Suppression of miR-142-3p reduces the release of IL-6in THP-1cells.