Regulatory Relationship Between LncRNA MALAT1 And MicroRNA181b And Possible Role In The Angiogenesis After Ischemic Stroke

In order to clarify the correlation between lnc RNA MALAT1 and mi RNA181 b,the computer biology software was used to predict the locus by "base complementary principle".The predicted sites were cloned into the double luciferase reporter gene for further validation.After confirmation of the loci,the correlation between the predicted expressions was further studied.In addition,mi RNA181 b could bind to the target gene m RNA-3’UTR of GRP78 to regulate the expression of GRP78.The expression level of GRP78 was detected by changing the concentration of lnc RNA MALAT1 and mi RNA181 b,and the relationship between the expression of GRP78 was clarified.Otherwise adjust the direction of the experiment.At present,there are human umbilical vein endothelial cells(HUVEC)and microvascular endothelial cells(MVEC).In this study,we selected mouse brain microvascular endothelial cells(b End3),the main reasons are: cell culture is simple,no special requirements on the culture medium;its rapid value,sensitive to vascular factors;source mice,in vitro and in vivo experimental phase convergence.A large number of studies have shown that: after ischemic stroke,VEGF,TNF-α,ANG-I and other factors increased significantly,and promote angiogenesis.Therefore,the expression of lnc RNA MALAT1 and mi RNA181 b was detected on the basis of observing cell migration and migration by stimulating the target cells after using the factors of one or more ischemic stroke to simulate the post-stroke high concentration factor environment.While using oxygen-glucose deprivation(OGD)to target the ischemic model of the cells to detect changes expression of lnc RNA MALAT1 and mi RNA181 b.Finally,the target cells were stimulated by changing the expression level of both,and the cell proliferation,migration,related angiogenic factors and target protein were detected.In the study of angiogenic factors,VEGF research is the most thorough.Its family members VEGFA and vascular neonatal most closely.VEGFA and VEGFR2 binding activation of two signal pathways:activation of Src signaling pathway,leading to endothelial cells on the α6β1 integrin and its ligand separation,and further activation of metal matrix protease(MMPs,mainly MMP2,MMP9),degradation of extracellular matrix,promote endothelial cell migration;Second,the activation of the classic PI3 / AKT signaling pathway,the release of calcium ions,PKC and other second messenger to promote endothelial cell activation,division,proliferation,promote angiogenesis.AKT and P-AKT are the core proteins of PI3 / AKT signaling pathway.Because VEGF is included in the experimental target,VEGF cannot be used as a stimulus.In addition,ANG-I must be combined with its receptor to play a role,but its receptor exists only in the damaged endothelial cell surface.At present,it is reported that TNF-α stimulates HUVEC to observe cell proliferation and mi RNA expression.Combined with laboratory conditions and experimental data,TNF-α is selected as the stimulating factor.The related test indicators and methods are as follows: angiogenesis: cell proliferation(MTT),cell migration(Transwell method);Overexpression of mi RNA181b(mi RNA 181 b mimics),inhibition of mi RNA181b(mi RNA 181 b inhibitor);Overexpressing lnc RNA MALAT1(plasmid),inhibiting lnc RNA MALAT1(si RNA or si MALAT1);The expression of VEGFA,MMP2 and MMP9 were detected by q-PCR.The expression of VEGF,AKT / P-AKT and GRP78 was detected by Western Blot.Part I: Prediction both locus and Detection of the correlation between lnc RNA MALAT1 and mi RNA181bObjective:To determine whether there are binding sites between lnc RNA MALAT1 and mi RNA181 b and the regulation relationship between them.Materials and Methods:First,mi RNA and lnc RNA biological database to find lnc RNA MALAT1,mi RNA181 b base sequence;The presence of binding sites between lnc RNA MALAT1 and mi RNA181 b was predicted by computer biological software ’RNA 22 version 2.0’.If present,the predicted site is cloned to the double luciferase reporter gene to further confirm the reliability of the site.Finally,the expression levels of lnc RNA MALAT1 、mi RNA181 b and GRP78 were detected by mi RNA181 bmimics / inhibitor and si MALAT1 transfected cells to clear the relationship between them.Results: There were two binding sites between the mouse lnc RNA MALAT1 and mi RNA181 b,and the left side of the locus was located at 2211,5124 position of the lnc RNA MALAT1 sequence.The double luciferase reporter gene system showed that the mouse mi RNA181 b could decrease the luciferin Enzyme expression;mi RNA181 b mimics inhibited the expression of lnc RNA MALAT1 and GRP78;mi RNA181 b inhibitor promoted the expression of lnc RNA MALAT1 and GRP78;si MALAT1 promoted the expression of mi RNA181 b but inhibited the expression of GRP78.Conclusions:The binding sites of MALAT1 and mi RNA181 b in mice were predicted by computer.Luciferase reporter gene further confirmed the relationship between the two;LncRNA MALAT1 and mi RNA181 b can negatively control each other.Part II: Expression of lnc RNA MALAT1 and mi RNA 181 b under hypoxia and hypoglycemiaAnd both of them on cell migration,VEGFA / MMP2 / MMP9 / VEGF / AKT / P-AKTObjective:Changes of expression of lncRNA MALAT1 and mi RNA181 b under TNF-α stimulation and hypoxia and hypoglycemia.And the expression of VEGFA / MMP2 / MMP9 / VEGF / AKT / P-AKT.Materials and Methods : First,different concentrations of TNF-α stimulated b End3 cells,at different time points to detect cell proliferation(MTT method)migration(Transwell method),explore the best TNF-α stimulation concentration.The expression levels of lnc RNA MALAT1 and mi RNA181 b were detected by q-PCR.The expression level of MALAT1 and mi RNA181 b was also detected by OGD-induced target cell ischemia model.The expression of VEGFA,MMP2,MMP9,AKT and P-AKT were detected by overexpression / inhibition of mi RNA181 b and inhibition of lnc RNA MALAT1 expression.Results:The proliferation of bEnd3 cells was almost the same at 10 ng / ml,20 ng / ml and 50 ng / ml.The proliferation of b End3 cells was not obvious,but the cell proliferation was significantly higher than that of the control group(P <0.001).Cells cultured for 24 hours the proliferation of cells significantly than other time points(P <0.05).10 ng / m L TNF-α concentration to stimulate b End3 cells,cultured 24 hours later,compared with the control group of cells migrate significantly;b End3 cells were cultured for 24 hours after 24 hours of OGD stimulation to promote the expression of mi RNA181 b and inhibit the expression of lnc RNA MALAT1.Q-PCR: MALAT1 expression decreased,mi RNA181 b expression increased,compared with the control group was significantly different(P were less than 0.05).Mi RNA181 b mimics / inhibitor was transfected into b End3 for 24 hours: mimics group promoted the proliferation and migration of VEGFA,MMP2,MMP9,VEGF,AKT and P-AKT.Inhibitor group,the expression of VEGFA,MMP2,MMP9,VEGF,AKT and P-AKT were inhibited.The expression of VEGFA,MMP2,MMP9,VEGF,AKT and P-AKT was inhibited by si MALAT1.Conclusions: After ischemic stroke,lnc RNAMALAT1 binds to mi RNA181 b and competes to regulate the function of mi RNA181 b.At the same time,mi RNA181 b can reverse the expression of MALAT1,promote the expression of VEGFA,AKT and P-AKT in endothelial cells,activate PI3 K / Akt signaling pathway.While activating Src signaling pathway,and promote local endothelial cell ring local α6β1 integrin increased to activate metal matrix protease MMPs(MMP2,MMP9),increased foot ring outward migration.Ultimately affect the proliferation of endothelial cells,migration,promote angiogenesis.