The Mechanism Study Of LonRNA TUG1 Contributes To Cerebral Ischemia/Reperfusion Injury

Part ? The role of lncRNATUG1 Protects the cerebral ischemia reperfusiosecondary n injuryBackground:Because of the improved living standard and aging population in recent years,cerebral ischemic stroke,with a rising morbidity,has become a main killer for peoples'death in our country.Clinically we found the damage to the brain more severe when get blood perfused again after the ischemic stroke.Some research indicates that the excessive inflammatory response in ischemic penumbra after the stroke takes the crucial responsibility for the secondary brain injury:while stimulating the anti-inflammatory signaling pathways would exacerbate the brain injury,and inhibiting inflammatory mediators from excessively expression can alleviate it.So it's a real challenge for us how to effectively prevent CRI(cerebral ischemia reperfusion injury).Constantly there are researches show lncRNA(long non-coding RNA)plays an important role in the regulation of the protection of cardiovascular and cerebrovascular diseases.Now by using astrocyte(AS)line OGD/RX(oxygen deficit/reoxygenation)models and MCAO(middle cerebral artery occlusion)models in mice,the aim of this experiment is to find out how lncRNA TUG1(taurine upregulated gene 1)protect the CRI.Method:By cultured the AS cells,we establish the OGD/RX model,and divide them into 4 groups:normal control,OGD 6h/RX 6h,OGD 6h/RX 24h,OGD 6h/RX 24h.The expression of IncRNA TUG1 was measured by RT-PCR(quantitative real-time polymerase chain reaction).The degree of cells damage was detected by testing the content of LDH(lactate dehydrogenase).The degree of apoptosis was evaluated using flow cytometric analysis.We compared the difference of the expression of IncRNA TUG1 in the four groups.We will further explored the role of CRI of the protective function of lncRNA TUG1.We use three different siRNA(small interfering RNA)to interfere with the expression of IncRNA TUG1.We testify if the brain protection of lncRNA TUG 1 remain when the function of lncRNA TUG1 is interfered.Results:lncRNA TUG1 expression has a significantly risen(P<0.05)after OGD/RX by RT-PCR.While LDH testing shows lncRNA TUG1 siRNA is protective toward the injury caused by OGD(P<0.05),flow cytometry testing reveals the apoptosis level has a significantly lessen after lncRNA TUG 1 siRNA interfering.Conclusions:IncRNA TUG1 play an important role in the AS cells undergoing OGD/RX.It can aggravate the apoptosis of cells.If the expression of lncRNA TUG1 inhibited,the number of apoptotic cells will deline.Part ? lncRNATUG1 Performs Its Function By Regulating AQP4 Objective:Since we discover in our previous research that AQP4 plays a crucial role in CRI,we make a prediction that IncRNA TUG1 may do its work by regulating AQP4.Method:concentrationWe used siRNA to interfere the expression of AQP4 in the line of AS.Then we bulid the mode of OGD/RX.We used untreated and TUG1 siRNA to treat the cells above.The cell damage is still exist or not in the cells above.We used RT-PCR AND WB(Western Blot)to detect the expression of AQP4 when the functiom of TUG1 is interfered by siTUG1.We detect the of LDH when the AQP4 siRNA interfered the function of AQP4.Flow cytometry was used to detect apoptosis.When the function of AQP4 is interfered,TUG1 further damage cells is still exsit or not?Results:WB testing shows the expression of AQP4 falls(P<0.05)after TUG 1 siRNA's interfering.When the function of AQP4 is interfered,TUG1 becomes ineffective by LDH testing(P<0.05),meanwhile it shows no further damage to the cell by flow cytometry testing.Conclusions:IncRNA TUG1 does the damage to the cell through the variation of AQP4's expression.Part ? Mechanisms of IncRNA TUG1 Regulating AQP4-by regulating of the expression of microRNA-145Objective:To detect whether TUG 1 regulates neuronal injury cells by regulating the expression of microRNA-145.Method:We used Starbase software to predict the binding sites of TUG 1 to microRNA-145.Realtime-PCR was used to detect the expression of microRNA-145 before and after TUG1 siRNA transfection.AS cells were treated with microRNA-145(mimics and inhibitor)and the expression of AQP4 was observed before and after treatment.AS were pretreated with microRNA-145 inhibitor,then treated with untreated and TUG1 siRNA-1(inhibitor-pretreated cells were cultured for 24 hours and then transfected with TUG1 siRNA for 6 hours).OGD/RX model was established to observe whether TUG1 siRNA had protective effect on cells.LDH detection,first treated with microRNA-145 inhibitor,OGD/RX model was established to observe whether the injury aggravated.After treatment with TUG1 siRNA,the protective effect of TUG1 siRNA was observed.Flow cytometry was used to detect apoptosis.After treatment with microRNA-145 inhibitor and then with TUG1 siRNA,the protective effect of siTUG1 existed.After successful interference with TUG1 siRNA 1,AS cells were treated with untreated and inhibitor(TUG1 siRNA pretreated cells were cultured for 6 hours and then treated with inhibitor for 6 hours).OGD/RX model was established to observe the protective effect of TUG 1 siRNA on cells.LDH test results showed that TUG1 interfered with the cell damage was reduced,and then the protective effect was observed with the effect of microRNA-145 inhibitor.Results:(1)Under normal conditions,WB results showed that microRNA-145 could mediate the regulation of AQP4 expression.When the expression of microRNA-145 increased,the expression of AQP4 decreased(P<0.001),When the expression of microRNA-145 was inhibited,the expression of AQP4 increased(P<0.001).(2)After OGD/RX,WB results showed that AQP4 expression in AS cells increased significantly compared with that in normal control group(P<0.01).However,the expression of AQP4 decreased significantly after the addition of microRNA-145 mimics(P<0.001).After inhibiting the effect of microRNA-145,the expression of AQP4 increased,even significantly higher than that after OGD/RX without intervention(P<0.01).(3)Normally,we showed that TUG1 could also mediate the expression of microRNA-145 by RT-PCR.When the role of TUG 1 was interfered,the expression of microRNA-145 increased(P<0.05).(4)LDH cytotoxicity assay showed that LDH content in cells after OGD was significantly higher than that in normal control group(P<0.01).However,after adding microRNA-145,the concentration of LDH in cells inhibit further compared with that of cells without adding OGD,and there was no significant change in LDH of cells after adding siTUG1.(5)Flow cytometry showed that compared with the normal control group,the apoptotic rate of cells increased significantly after OGD/RX treatment(P<0.001).After OGD/RX+microRNA-145 inhibitor treatment,the apoptotic rate of cells increased further(P<0.01).The apoptotic rate of OGD/RX cells added with miR-145 inhibitor and siTUG did not change significantly.However,if OGD/RX cells were not added with microRNA-145 inhibitor,the apoptotic rate of cells decreased slightly after adding siTUG1(P<0.01).Conclusions:The expression of IncRNA TUG1 and AQP4 mediates the damage and apoptosis of AS cells after OGD/RX through the regulation of microRNA-145.Part IV In vivo experiments confirmed that lncRNA TUG1 is involved in regulating CRI damageObjective:MCAO experiments were conducted in C57/BL6 male mice to simulate cerebral ischemia-reperfusion injury and to observe whether TUG1 participates in CRI.Method:C57/BL6 male mice were divided into sham operation group,MCAO group,MCAO+Negative shRNA group and MCAO+TUG 1 shRNA group.The area of cerebral infarction area,neurological score and TUNEL staining were used to confirm whether TUG1 was involved in regulating cerebral ischemia,hypoxia and reperfusion injury.Results:(1)The infarct size increased after MCAO in mice,but did not change significantly after injecting Negative shRNA into lateral ventricle,but decreased significantly after injecting TUG1 shRNA into lateral ventricle(P<0.01).(2)After MCAO in mice,Bederson score increased significantly compared with sham-operated group(P<0.001).After lateral ventricle Negative shRNA injection,there was no significant difference in bederson score between MCAO group and MCAO group(P<0.01).(3)Compared with sham-operated group,the number of apoptotic cells increased significantly after MCAO in mice(P<0.01).After lateral ventricle Negative shRNA injection,there was no significant difference in apoptotic number between MCAO group and MCAO group.After lateral ventricle TUG1 shRNA injection,the number of apoptotic cells was significantly decreased(P<0.001).Conclusions:When CRI occurs,TUG 1 can protect AS cells from ischemia,hypoxia and reperfusion injury when its role is disrupted.