The Effect Of MiR-155 On Cerebral Ischemia Reperfusion Injury By Targeting MafB

Cerebral ischemia reperfusion injury(CIRI)is ischemic cerebral vascular disease,which is mainly caused by prolonged ischemia and aggravation of ischemic injury by re-circulation of blood vessels.During CIRI,the production of reactive oxygen species can cause oxidative damage of brain lipids,proteins and DNA,leading to neurological dysfunction and cell death.Treating CIRI remains a challenge for clinicians.The pathogenesis of CIRI is very complex,and it is of great significance to study the function of its related gene.Micrornas(miRNAs)are a group of conserved,small,non-coding RNAs,which are usually 18 to 25 nucleotides in length,and can regulate the expression of target genes.Some studies have confirmed that miR-155 knockout can protect lps-induced microglial BV2 inflammatory injury by regulating RACK1 expression and activating the MAPK/NF-κB and mTOR signaling pathway.Microglia cells can produce inflammatory mediators and neurotoxic compounds,such as IL-1β,IL-6,TNF-α,reactive oxygen species,nitric oxide and prostaglandin E2,which are important determinants of neuronal death in cerebral ischemia.Therefore,miR-155 may play an important role in CIRI.MafB,a member of the MAF transcription factor family,is widely expressed in human tissues,but is particularly abundant in myeloid cells,which contribut to the establishment and maintenance of monocytes and macrophages.Preliminary bioinformatics prediction showed that MafB was the target gene of miR-155.In addition,studies have shown that miR-155-5p can promote the expression of IL-6 and GLP by specifically inhibiting the expression of MafB,thereby reducing blood glucose level.However,the specific role of miR-155 in CIRI by targeting MafB has not been studied.SH-SY5Y cells are human neuroblastoma cells with similar morphology,neurochemical and electrophysiological characteristics to neurons,which have been widely used as in vitro models for studying neuron injury or death.In this study,the effect of miR-155 on CIRI by targeting MafB were studied.Firstly,qRT-PCR was carried out to detect the expression of miR-155 and MafB in plasma of normal human and CIRI patients,and GraphPad Prism 7 was performed to analyze the correlation between the miR-155 and MafB expression in plasma of CIRI patients.In vitro,miR-155 overexpression and knockout vectors were constructed,and the effect of miR-155 overexpression and knockout on proliferation,apoptosis,invasion,migration,cytokine secretion and MafB expression of OGD/SH-SY5Y cells were investigated by using SH-SY5Y cells as OGD/R deprivation and reoxygenation(OGD/R)model.Bioinformatics was also performed to analyze the relationship between miR-155 and MafB,and MafB knockout vector was established,and the effect of miR-155 on the proliferation,apoptosis,invasion,migration and cytokine secretion of OGD/R SH-SY5Y cells by targeting MafB was explored.Finally in vivo study,mouse ischemia-reperfusion injury model(MCAO)was established by the method of line switch,the lateral ventricle was injected with miR-155 mimic and miR-155 antagomir;TTC assay,immunohistochemistry assay,Western blot and ELISA assay were performed to measure the effect of miR-155 on cerebral infarction volume,MafB,IL-1β,IL-6,TNF-α,iNOS and COX-2 levels in brain tissues of MCAO mice,investigating the effect of miR-155 on CIRI model in mice by targeting MafB.This study is divided into the following three parts:Part Ⅰ:Expression of miR-155 and MafB in plasma of patients with CIRI;Part Ⅱ:Effect of miR-155 on ischemia reperfusion injury model by targeting MafB in vitro;Part Ⅲ:Effect of miR-155 on CIRI model by targeting MafB in mice.Main content:Part Ⅰ:Expression of miR-155 and MafB in plasma of patients with CIRIMethods1.The expression of miR-155 and MafB in plasma of normal subjects and patients with CIRI was dermined by qRT-PCR.2.GraphPad Prism 7 was carried out to analyze the correlation between the expression of miR-155 and MafB in plasma of patients with CIRI.ResultsCompared with normal human,the expression of miR-155 in plasma of patients with CIRI was significantly increased,while the expression of MafB was significantly decreased,and the expression of miR-155 were negatively correlated with MafB.Part II:Effect of miR-155 on ischemia reperfusion injury model by targeting MafB in vitroMethods1.Establish OGD/R SH-SY5Y models,and the expression of miR-155 and MafB in OGD/R SH-SY5Y cells was measured by qRT-PCR.2.miR-155 overexpressed and interfered lentivirus vectors were constructed,and the recombinant lentivirus infected SH-SY5Y and OGD/R SH-SY5Y cells after packaging and titration to obtain stable cell lines.The expression of miR-155 and MafB in OGD/R SH-SY5Y cells in each group were detected by qRT-PCR.3.MTT assay was performed to determin the proliferation of cells treated with different treatments.4.Flow cytometry assay was implemented to detect the apoptosis of SH-SY5Y cells in different treatment groups.5.Transwell assay was carried out to test the invasion of SH-SY5Y cells in different treatment groups.6.The scratch assay was performed to determin the migration of SH-SY5Y cells in different treatment groups.7.The level of IL-1β,IL-6 and TNF-α in the supernatant of SH-SY5Y cells cultured in different treatment groups were measured by enzyme-linked immunosorbent assay(ELISA).8.The relationship between miR-155 and MafB was verified by the dual luciferase reporter gene assay.9.Western blot was carried out to detect the expression of MafB,iNOS and COX-2 proteins in SH-SY5Y cells of different treatment groups.Results1.Compared with control cells,miR-155 expression was significantly increased and MafB expression was significantly decreased in SH-SY5Y cells treated with OGD/R.2.The overexpression of miR-155 significantly inhibited the proliferation,invasion and migration of OGD/R SH-SY5Y cells,promoted cell apoptosis,and increased the levels of IL-1β,IL-6 and TNF-α in the superstratum of OGD/R SH-SY5Y cells,as well as the expression of iNOS and COX-2 in the OGD/R SH-SY5Y cells.However,miR-155 knockout significantly promoted the proliferation,invasion and migration of OGD/R SH-SY5Y cells,inhibited cell apoptosis,and reduced the levels of IL-1β,IL-6 and TNF-α in the superstratum of OGD/R SH-SY5Y cells,as well as the expression of iNOS and COX-2 in the OGD/R SH-SY5Y cells.3.miR-155 could inhibit the proliferation,invasion and migration of OGD/R SH-SY5Y cells,promote cells apoptosis,and increase the levels of IL-1β IL-6 and TNF-α in the superstratum of OGD/R SH-SY5Y cells,as well as the expression of iNOS and COX-2 in the OGD/R SH-SY5Y cells by targeting MafB.Part Ⅲ:Effect of miR-155 on CIRI model by targeting MafB in mice Methods1.MCAO model was constructed with the method of wire bolting,lateral ventricles of mice were injected with miR-155 mimic and miR-155 antagomir,and the cerebral infarction volume of MCAO mice in different treatment groups was measured by TTC method.2.Western blot and immunohistochemistry assay were used to detect the effect of miR-155 on the expression of MafB protein in the brain tissues of MCAO mice.3.qRT-PCR was used to detect the expression of miR-155,iNOS and COX-2 in the brain tissues of MCAO mice in each group.4.The levels of IL-1β,IL-6 and TNF-α in the brain tissues of MCAO mice were dermined by ELISA assay.5.Western blot was used to detect the expression of iNOS and COX-2 proteins in the brain tissues of MCAO mice.ResultsStudies based on MCAO model demonstrated that miR-155 might increase the volume of cerebral infarction,promote the levels of IL-1β,IL-6,TNF-α,iNOS and COX-2 by targeting MafB,thereby promoting the inflammatory response caused by MCAO and increasing the brain injury.Conclusion1.In plasma of patients with CIRI,the expression of miR-155 was significantly increased,while that of MafB was significantly decreased,and the expression of miR-155 were negatively correlated with MafB.2.miR-155 could inhibit the proliferation,invasion and migration of OGD/R SH-SY5Y cells,promote cells apoptosis,and increase the levels of IL-1β,IL-6 and TNF-α in the superfloant of OGD/R SH-SY5Y cells,as well as the expression of iNOS and COX-2 in the OGD/R SH-SY5Y cells by targeting MafB,ultimately exacerbating nerve cell damage and inflammatory responses.3.Studies by MCAO model exhibited that miR-155 might increase the area of cerebral infarction,promote the levels of IL-1β,IL-6,TNF-α,iNOS and COX-2 by targeting MafB,thereby promoting the inflammatory response caused by MCAO and increasing the brain injury.