The Role Of TLR4/miR-181a Axis In Brain Injuries After Cerebral Ischemia/Repefrusion And Its Mechanism

TLR4/microRNA axis was considered as a novel signal transduction pathways, widelyinvolved in various pathological and physiological process, and thus therapies targeted theaxis had enormous clinical application prospect. In this study, we aimed to investigate theregulatory effect of TLR4/microRNA axis on nerve injuries and its specific mechanism inmicroglia and cerebral ischemia reperfusion mice model using bioinformatics, immunologyand molecular biology method. This research could be divided into three parts:1. TLR4activation increased miR-181a via AP1in microglial cells.Previous studies had proved that TLR4activation could regulate microRNA expressionvia the AP1, NF-kappa B and IRF. In this part, we stimulated microglia with TLR4agonistEc-LPS, and detected the microRNA profile related to TLR4activation using DeepSequencing. Results showed that TLR4activation upregulated43microRNAs more than4times and downregulated27microRNAs more than four times. qRT-PCR verification showedthat miR-181a expression rised significantly in microglial cells after TLR4activation.Through a database query, we found that there was an AP1binding site on the1976bpupstream of the miR-181a gene. Then We cloned miR-181a promoter into dual luciferasereporter and co-transfected it into HEK-293cells with AP1overexpression vector. The resultsindicated that AP1could bind to miR-181a promoter and regulate its gene transcription.Morevover, AP1knock-down could block the upregulation of miR-181a induced by TLR4activation in microglial cells. Furthermore, we found that transfection of miR-181a inhibitorreduced the synthesis and release of IL-1beta and TNF-alpha from microglia stimulated byTLR4agonist. These results suggested that TLR4upregulated miR-181a expression in microglia via AP1, and miR-181a could play an important role in proinflammatory effects ofTLR4activation.2. miR-181a aggravated inflammatory response and apoptosis after cerebralischemia/reperfusionThe results of the first parts suggested miR-181a played an important role in TLR4proinflammatory effects. In this part, we firstly detected miR-181a expression in the brains ofnormal mice and the cerebral ischemia/reperfusion model, and the result showed thatmiR-181a upregulated after ischemia/reperfusion. Furthermore, in order to investigate theeffect of miR-181a on nerve injury in vivo, we constructed an overexpression AAV9plasmidof miR-181a and injected it through the tail vein. We demonstrated that the AAV9plasmidtransfected into mice could be detected in the cerebral cortex and hippocampus at a high leveland overexpression of miR-181a could aggravate histopathologic changes, expand infarctionareas and increase the neurological deficit scores of mice models. Moreover, levels ofinflammatory factor IL-1beta and TNF-alpha in brain tissues of mice transfected withmiR-181a were higher than those of normal mice, as well as the pecentage of Tunel andCaspase3positive cells. The results suggested the miR-181a expression upregulated aftercerebral ischemia/reperfusion, and promoted reperfusion inflammatory reaction and apoptosis,and thus aggravated nerve damage.3. miR-181a inhibits TGF-beta/SMADs signaling via targeting TGFBR1Part1and Part2have showed that TLR4increased miR-181a expression in microglialcells via AP1, and miR-181a could promote inflammation and apoptosis after cerebralischemia reperfusion, and thus increased neurological deficit. So TLR4and miR-181aformed an axis, regulating nerve injury reactions after cerebral ischemia reperfusion.However, the downstream targets and specific regulatory mechanism of TLR4/miR-181aaxis still need to be clarified. In this part, we searched through bioinformatics database formiR-181a downstream targets and found900possible target genes, including TGFBR1,receptor of the important neuroprotective factor TGF-beta. So we focused on TGFBR1. Result of western blot confirmed that miR-181a mimic downregulated TGFBR1proteinexpression in BV2cells, whereas miR-181a inhibitor could elevate its protein levels. We alsofound that miR-181a had no obvious effect on TGFBR1mRNA expression. Furthermore,When a wild type TGFBR13’-UTR luciferase reporterwas co-transfected with miR-181amimics into HEK-293cells, therelative luciferase activity was significantly reducedcompared with that of the negative control, whereas mutationof one sequence or the otherattenuated the repression mediatedby the miR-18b mimics. In contrast, the luciferase activityin cellstransfected with empty vector or the reporter carrying both mutant sequences was notaffected. We also showed that miR-181a plasmid injection could decrease TGFBR1level inthe brain tissues of cerebral ischemia/reperfusion mice,as well as its downstream moleculesp-SMAD2and p-SMAD3. These results fully demonstrated miR-181a could regulateTGFBR1expression at the post-transcriptional level and inhibits TGF-beta1/SMADssignal transduction/after cerebral ischemia reperfusion. Combining with the former two parts,we showed that TLR4and miR-181a formed an axis, which targeted TGF-beta1/SMADssignaling and thus aggravated the inflammation and apopotosis after cerebralischemia/reperfusion.