| Objectives:To screen and select a targeted toll like receptor4gene(TLR4mRNA)microRNA(miRNA/miR) and observe the effect of the selected miRNA on mediatorsof inflammation expression in macrophage and investigate its molecular mechanisms.Methods:Bioinformatics approach was used to analyze the secondary structure ofthe sequence of3′Untranslated Regions(3′UTR) of TLR4mRNA, meanwhile amiRNA which possibly targeted TLR4gene was selected by online predictionsoftware together with pertinent literature. The contents of TNF-α and IL-1β and IL-6in culture medium were tested by enzyme linked immunosorbent assay(ELISA). Therelative level of the selected miRNA intra-cellular was detected by real timequantitative polymerase chain reaction(qPCR). The relative expressions of TLR4mRNA and TNF-α mRNA and IL-1β mRNA together with IL-6mRNA intra-cellularwere measured by reverse transcription polymerase chain reaction(RT-PCR). Thelevel of TLR4protein intra-cellular was determined by western blot. The luciferasereporter genetic technology was used to survey the activity of the selected miRNAbinding to TLR4mRNA. All experimental data were demonstrated by mean±standarddeviation (X±S). Statistical analysis results and graphs were generated by GraphpadPrism v5.0. P<0.05was considered significant.Results:⑴MiR-181a which probably targeted TLR4gene(TLR4mRNA)wasselected as guarantined miRNA.⑵When miR-181a mimic control or miR-181a mimicwith different concentration(2.5nM or5.0nM or10.0nM)was transfected into THP-1macrophages for0h or24h or48h or72h, then the contents of TNF-α and IL-1β andIL-6in culture medium were tested after the cells were separately stimulated to LPS for6h, we observed that of each miR-181a mimic transfection group was significantlylower compared to the control or0h group(all P＜0.05)and the condition of the5.0nMmatching48h group was the most predominant among overall groups. However, whenmiR-181a inhibitor control or miR-181a inhibitor with different concentration(2.5nMor5.0nM or10.0nM)was transfected into them for0h or24h or48h or72h, then theidentical indexes in culture medium were assayed after they were uniformly treated toLPS for6h, we detected that of each miR-181a inhibitor transfection group wasobviously higher compared to its control or0h group(all P＜0.05)and the condition ofthe5.0nM matching48h group was the most notable among them.⑶After miR-181amimic or its control(5.0nM)and miR-181a inhibitor or the control(5.0nM)wererespectively transfected into THP-1macrophages for48h, then the cells werestimulated to LPS for6h, consequently, the relative quantity of miR-181aintra-cellular increased significantly when the one of miR-181a mimic transfectiongroup was compared to the control(P＜0.05)but the one decreased notably when thatof miR-181a inhibitor transfection group was compared to its control(P＜0.05),meanwhile, no significant difference from TLR4mRNA relative expressionintra-cellular was discovered either between miR-181a mimic transfection group andits control(P＞0.05)or miR-181a inhibitor group and the control(all P＞0.05),however, the levels of TLR4protein and TNF-α mRNA and IL-1β mRNA as well asIL-6mRNA intra-cellular of miR-181a mimic transfection group were significantlylower than that of the control(all P＜0.05)but the one of miR-181a inhibitor groupwere obviously higher than that of its control(all P＜0.05).⑷From the luciferasereporter genetic technology, we observed that the activity of luciferase in293T cellsof miR-181a mimic transfection group was apparently lower than that of the control(P＜0.05)but the one of miR-181a mimic and miR-181a inhibitor cotransfection groupwas significantly higher than that of miR-181a mimic group(P＜0.05).Conclusions:①Hsa-miR-181a might negatively regulate mediators ofinflammation expression in THP-1macrophage via down-regulating TLR4expression.②The mechanism of hsa-miR-181a suppressing TLR4gene in THP-1macrophage is that hsa-miR-181a could directly bind to the3′UTR of TLR4mRNA and thenrepresses the translation of TLR4mRNA. |
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