Downregulation Of The Long Non-coding RNA H19 Inhibits Angiogenesis After Ischemic Brain Injury By Inducing Autophagy

Objective: Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone.Strategies to improve angiogenesis may facilitate the function recovery after stroke.Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis,but less is known about the role of lncRNAs in regulating cerebral ischemia-induced angiogenesis.In this study,we aim to investigate the expression profile of lncRNAs in post-stroke mice and the effects of lncRNA H19 on the biological behavior of hCMEC/D3 s cells under oxygen-glucose deprivation(OGD)and the related mechanisms.Methods:We established male C57BL/6 mouse MCAO model with the electrocoagulation method.TTC staining and 2-dimensional laser speckle were used to assess the model.Subsequently,the expression profiles of lncRNAs in ischemic penumbra and brain tissue of the sham mice were established by the SBC microarray.The microarray results were confirmed by qRT-PCR randomly and the lncRNA H19 was selected to be further studied.The H19 expressions in mice ischemic penumbra and human cerebral microvascular endothelial cells under oxygen-glucose deprivation were observed dynamically.After H19 was knocked down by siRNA,we performed CCK-8 assay,transwell assay,tube formation assay and flow cytometry to detect the effects on endothelial cells behavior.We further examined the expression changes of autophagy-related protein after knockdown of H19.The results indicated that down-regulation of H19 might inhibit angiogenesis by inducing autophagy.Results:1.Successful establishment of C57BL/6 MCAO model.2.The expression of lncRNAs in ischemic brain tissue changed significantly after stroke,and 58952 lncRNAs were detected in the mouse SBC microarray after cerebral ischemia.Compared with the sham group,234 lncRNAs were up-regulated more than 2-fold and P<0.05,and 50 lncRNAs were down-regulated more than 0.5-fold.Eight lncRNAs were validated by qRT-PCR,which were consistent with the results of the SBC microarray.The expression profiles of lncRNAs in cerebral ischemia were successfully established and lncRNA H19 was selected for the subsequent studies.3.The results of qRT-PCR showed that the expression of H19 in dMCAO mice and OGD 3h cells increased dynamically compared with the control group;4.H19 siRNA was picked up and successfully knocked down the lncRNA H19 of hCMEC/D3 cells.SiH19 inhibited the proliferation,migration and tube formation of hCMEC/D3,and promoted the apoptosis process of hCMEC / D3 cells;5.The expression of VEGF and FGF2 decreased after H19 knockdown;6.The expression level of autophagy-related proteins increased after H19 knockdown,indicating that knockdown of H19 may inhibit angiogenesis by activating autophagy levels.Conclusions:1.The expression profile of lncRNAs in ischemic brain tissue changed significantly after ischemic stroke,and lncRNAs were involved in the angiogenesis process after stroke.2.LncRNA H19 knockdown inhibited the proliferation,migration and formation of microvascular endothelial cells,promoted apoptosis of endothelial cells,and then affected angiogenesis after stroke.The mechanism may be activation of autophagy pathway.