LncRNA-MEG3 Regulates Microglial Polarization Through KLF4 To Affect Cerebral Ischemia-reperfusion Injury

Cerebral ischemia-reperfusion injury（CIRI）refers to the recovery of blood after a period of cerebral ischemia,which will trigger a series of pathological reactions and cause more serious secondary damage.After reperfusion,the biomolecules in cells and tissues are damaged.The pathogenesis of ischemia-reperfusion（I/R）injury is multifactorial and complex,involving inflammation,calcium overload,oxidative/nitrosative stress,endoplasmic reticulum stress,epigenetic changes,etc.Microglia,as immune effector cells residing in the central nervous system,is the first and most important line of defense in the central nervous system.In the inflammatory response,microglia will undergo different forms of polarization activation,mainly including the polarization of M1 and M2.Some studies have shown that the conversion of M1 to M2 may be a potential treatment strategy for brain I/R injury.Microglia/macrophage polarization dynamics reveal a new mechanism of damage expansion after focal cerebral ischemia.Lnc RNA（long non-coding RNA）refers to non-coding RNA（nc RNA）with a length of more than 200 nucleotides,which affects normal biological processes and the progress of various diseases through a variety of regulatory mechanisms.Lnc RNA maternally expressed gene 3（lnc RNA-MEG3）has been shown to be involved in a variety of disease processes,such as a variety of cancers and osteoarthritis.The potential application of lnc RNA-MEG3 in cancer diagnosis and prognosis lnc RNA-MEG3 inhibits the proliferation and metastasis of gastric cancer through the p53 signaling pathway.In addition,lnc RNA-MEG3 regulates the inflammatory response of macrophages by regulating the expression of inflammatory factors.By regulating the lnc RNA-MEG3/mi R-204/CDKN2A regulatory axis,silencing lnc RNA-MEG3 can inhibit ox-LDL-induced macrophage inflammation and apoptosis.However,there are few reports on whether lnc RNA-MEG3 affects brain I/R damage by regulating microglia polarization.Inflammation is an important pathophysiological mechanism of CIRI and is involved in its occurrence and development.Inflammatory cells mainly include white blood cells,microglia and astrocytes.Microglia are resident immune cells in the central nervous system（CNS）,and are responsible for homeostasis.Many reactions during CIRI are associated with the activity of microglia and innate immune system cells.Therefore,further studies for microglia activity in the progress of CIRI will help to resist any brain injury after I/R injury.In addition,sample Kruppel factor 4（KLF4）can be applied to a variety of signaling pathways,thus have anti-inflammatory and proinflammatory effect,but KLF4 function in different periods of inflammatory response is different,and the mechanism of KLF4 also has difference in different cells and organization.Further researches on the role of KLF4in CIRI are of great significance for exploring the pathogenesis of CIRI,and provide new therapeutic targets for the treatment of CIRI.In this study,we will study the role of lnc RNA-MEG3 in cerebral I/R injury.At the same time,we will further clarify the specific molecular mechanism of lnc RNA-MEG3 regulating the secretion of inflammatory factors in microglia through in vivo and in vitro experiments.Materials and Methods（1）MCAO/R（middle cerebral artery occlusion/reperfusion）was used to induce CIRI mouse model.The mice were anesthetized with sodium pentobarbital.A 4-0surgical nylon wire with a blunt tip（0.23 mm in diameter）was inserted into the internal carotid artery to occlude the middle cerebral artery（MCA）.After 60minutes of proximal MCA occlusion,blood flow was restored by removing the suture（reperfusion）.For the sham operation group（n=8）,except for MCAO,the same anesthesia and surgical procedures were performed.The mice were sacrificed by reperfusion at 1d（n=8）,3d（n=6）,5d（n=6）,7d（n=6）and 14d（n=6）.Among them,two sham mice and two mice perfused for 1 day were stained with TTC to measure the cerebral infarct area of the mice,and 3 mice in each group were stained with immunofluorescence to detect the co-localization of CD86 or CD206with Iba1.Western blot and real-time quantitative PCR were performed on 3 mice to detect the levels of M1 labeled i NOS and M2 labeled Arg1,respectively.Mice were transfected and 24 mice were divided into four groups:sham,MCAO,MCAO+si-NC and MCAO+si-MEG3（n=6）.Mice in the MCAO group were treated with MCAO for 1 hour,and then perfused for 7 days.MCAO+si-NC or MCAO+si-MEG3 group:transfection was performed before MCAO treatment.According to the instructions,we mixed the mixture of si-NC or si-MEG3（100μmol/L）and Lipofectamine RNAi MAX transfection reagent（Invitrogen,Carlsbad,CA,USA）,and they were then incubated at room temperature for 20 minutes and injected into the right ventricle of the mouse before MCAO.After treatment,the brain tissues were removed.The neurological condition of the mice was evaluated by the modified nervous system severity score（m NSS）test.Real-time quantitative PCR was used to detect the level of lnc RNA-MEG3.Western blot detection includes the expression levels of KLF4,i NOS,and Arg1.The ELISA method was used to detect the levels of inflammatory factors TNF-α,IL-1β,IL-10 and IL-4 in the cell supernatant.（2）OGD/R（oxygen and glucose deprivation/reoxygenation）treatment:the medium of mouse microglia cell line BV2 cells was replaced with glucose-free DMEM,and kept in an anaerobic incubator with 95%N2 and 5%CO₂at 37°C for 2h to simulate OGD injury.Then the medium of BV2 cells was replaced with normal culture,and placed in a humid incubator at 37°C（95%air and 5%CO₂）.The cells were collected at different times（12,24,48 hours）.BV2 cells cultured without treatment were used as controls.Transfection group:control,OGD/R,OGD/R+si-NC and OGD/R+si-MEG3,si-NC and si-MEG3 were transfected into BV2 cells,and treated the cells with OGD for 2 h,and then perfused for 48 h.Real-time quantitative PCR was used to detect the expression of lnc RNA-MEG3.Western blot was used to detect the expression levels of CD86,i NOS,CD206,and Arg1.The ELISA method was used to detect the levels of inflammatory factors IL-1β,TNF-α,IL-4 and IL-10 in the cell supernatant.The lnc RNA-MEG3 and KLF4 overexpression plasmids were transfected or co-transfected into OGD/R-induced BV2 cells.Western blot was used to detect the expression levels of KLF4,CD86,i NOS,CD206,and Arg1.The ELISA was used to detect the levels of inflammatory factors TNF-α,IL-1β,IL-10 and IL-4 in the cell supernatant.（3）RNA pull-down and RNA immunoprecipitation（RIP）experiments were used to verify the binding of lnc RNA-MEG3 and KLF4 in BV2 cells.The lnc RNA-MEG3 overexpression plasmid pc DNA3.1-MEG3 or small interfering RNA si-MEG3 was transfected into BV2 cells,and then the levels of lnc RNA-MEG3 and KLF4 in the two groups of BV2 cells were detected by Western blot.Results（1）Compared with the Sham group,the brain tissue of the MCAO group showed a white infarct area.Mice were treated with MCAO for 1 hour and then reperfused at different times.Compared with the Sham group,the MCAO/R group mice had severe neurological dysfunction,but it gradually decreased with the increase of reperfusion time（P<0.01）,indicating the successful establishment of the mouse brain I/R model.In addition,compared with the Sham group,the lnc RNA-MEG3 level in the cerebral cortex infarcted area of MCAO/R mice after ischemia was significantly increased and continued to increase for at least 7 days（P<0.05）.MCAO/R mice showed significantly higher M1 marker i NOS levels in the infarcted cerebral cortex,and continued to increase for at least 7 days after ischemia（P<0.05）.On the contrary,3 days after reperfusion,the level of M2 marker Arg1continued to increase,but then gradually decreased（P<0.05）.After lnc RNA-MEG3 expression was knocked down,the m NSS score of MCAO/R treated mice was decreased.After knocking down lnc RNA-MEG3,the level of M1-labeled i NOS was significantly reduced in MCAO/R-treated mice,while KLF4 and M2-labeled Arg1 were elevated in MCAO/R-treated mice.Compared with MCAO/R-treated mice,the co-localization of Iba1 and CD86 was decreased after lnc RNA-MEG3 knockdown,while the co-localization of Iba1 and CD206 was increased.In addition,after knocking down lnc RNA-MEG3,the levels of pro-inflammatory factors TNF-αand IL-1βin MCAO/R brain tissues were decreased,while the levels of anti-inflammatory factors IL-10 and IL-4 were increased.（2）After BV2 cells were treated with OGD/R for 2 h,lnc RNA-MEG3 levels were increased with time for at least 48 hours.In the BV2 cells treated with OGD/R,the levels of M1 labeled CD86 and i NOS were significantly increased,while the levels of M2 labeled CD206 and Arg1 were significantly decreased.However,the transfection of si-MEG3 reversed these results.In BV2 cells treated with OGD/R,the levels of pro-inflammatory factors TNF-αand IL-1βwere increased,while the levels of anti-inflammatory factors IL-10 and IL-4 were decreased,while the transfection of si-MEG3 reversed these results.We transfected or co-transfected the lnc RNA-MEG3 and KLF4overexpression plasmids into BV2 cells induced by OGD/R,and the results showed that the overexpression of lnc RNA-MEG3 significantly increased the levels of CD86,i NOS,TNF-αand IL-1β,while significantly reduced the levels of CD206,Arg1,IL-10 and IL-4.In contrast,the overexpression of KLF4 significantly reduced the levels of CD86,i NOS,TNF-αand IL-1β,while significantly increased the levels of CD206,Arg1,IL-10 and IL-4.The overexpression of KLF4 reversed the effect of lnc RNA-MEG3 overexpression on the M1 and M2 markers and inflammatory factors in BV2 cells induced by OGD/R transfected with pc DNA-MEG3.（3）Lnc RNA-MEG3 was enriched in the KLF4 immunoprecipitation,while KLF4 protein was enriched in the lnc RNA-MEG3 pulled down complex,indicating that lnc RNA-MEG3 could bind to KLF4 in BV2 cells.After the transfection of pc DNA3.1-MEG3 in BV2 cells,the level of lnc RNA-MEG3 was significantly increased and the level of KLF4 protein was significantly decreased.On the contrary,after the transfection with si-MEG3,the lnc RNA-MEG3 level was significantly reduced,while the KLF4 protein level was significantly increased.These results proved that lnc RNA-MEG3 could bind to KLF4 and inhibited its expression.Conclusions（1）The expression level of lnc RNA-MEG3 in the brain tissues of MCAO/R mice was significantly up-regulated.At the same time,the level of M1 labeled i NOS increased,and the level of M2 labeled Arg1 continued to increase,but then gradually decreased.After knocking down lnc RNA-MEG3,the levels of pro-inflammatory factors TNF-αand IL-1βin the brain tissues of MCAO/R mice were decreased,while the levels of anti-inflammatory factors IL-10 and IL-4 were increased.The colocalization of Iba1 and CD86 was decreased,while the colocalization of Iba1 and CD206 was increased,indicating that lnc RNA-MEG3 might participate in the development of the polarization of mouse microglia from M1 to M2 by regulating inflammation.（2）After OGD/R treatment of BV2 cells,lnc RNA-MEG3 overexpression significantly increased the levels of CD86,i NOS,TNF-αand IL-1β,and significantly reduced CD206,Arg1,IL-10 and IL-4 levels.KLF4 overexpression reversed the effects of lnc RNA-MEG3 overexpression on the M1 and M2 markers and inflammatory factors in OGD/R-induced BV2 cells transfected with pc DNA-MEG3,indicating that KLF4 was involved in lnc RNA-MEG3-mediated OGD/R induced polarization of microglia.（3）Lnc RNA-MEG3 could bind to KLF4 protein in mouse microglia,and lnc RNA-MEG3 could bind to KLF4 and inhibited its expression.