Effect Of MiR-126 Transfected Bone Marrow Mesenchymal Stem Cells On Brain Microvascular Endothelial Cells Under Simulated Intracerebral Hemorrhage Condition

Objective:Intracranial hemorrhage（ICH）refers to the non-traumatic intracerebral hemorrhage in the brain,which is a serious type of cerebrovascular disease with a high morbidity,mortality and disability rate,accounting for about 20%-30%of all strokes.At present,neurology provides only symptomatic and supportive treatment for patients with ICH.Transplantation of bone marrow mesenchymal stem cells（BMSCs）transfected with micro RNAs（miRNAs）is a new method of treatment of diseases that has drawn much attention in recent years.BMSCs are multipotent stem cells that are easy to be obtained and cultured,have low immunogenicity,proliferate easily and migrate autonomously to the lesion area,making them an ideal therapeutic vector.In addition,many studies at home and abroad have proved that BMSCs can improve neurological deficits in experimental intracerebral hemorrhage rats through various mechanisms.miR-126 is a miRNA that is specifically distributed on the vascular endothelium and plays an important role in angiogenesis.Our previous study found that the level of miR-126 was significantly down-regulated in peripheral blood of patients with ICH and was positively correlated with the degree of edema in the tissue around the hematoma.Another study has found that miR-126 in experimental cerebral hemorrhage rats showed protective effect.Therefore,this study aims at our previous research results,combined with the fact that BMSCs can improve neurological deficits in experimental intracerebral hemorrhage rats,to explore the role of miR-126 transfected BMSCs in cerebral hemorrhage in vitro model.We hope that our study can provide a new focus for the treatment of cerebral hemorrhage.Methods:First,the synthetic rno-miR-126a-5p mimics,inhibitors and negative control were transfected into BMSCs,and then an intracerebral hemorrhage in vitro model was simulated to some extent by oxygen glucose deprivation（OGD）combined with Hemin.The transfection of gene was detected by real-time fluorescence quantitative PCR（RT-PCR）,and the expression of AKT\ERK1 was further detected by RT-PCR and western blot.Finally,CCK-8 was used to detect the proliferation of rat brain microvascular endothelial cells（RBMVECs）co-cultured with BMSCs under the condition of intracerebral hemorrhage in vitro model.Results:1.RT-PCR was performed on cells transfected with Negative Control（NC group）,cells transfected with rno-miR-126a-5P mimics（M group）and cells transfected with rno-miR-126a-5P inhibitors（I group）.PCR assay of gene transfection found that miR-126 expression of M group cells was significantly increased,and the difference was statistically significant（P<0.05）,prompted the successful transfection of miR-126 to BMSCs.2.The conditions of simulated cerebral hemorrhage in vitro:After transfection for 24-48h,cells were cultured with glucose-free and treated with 5%CO₂+0.4%O₂+95%N₂ for4h,10uM Hemin was added at the same time.To a certain extent,we simulated cerebral hemorrhage in vitro model.3.The mRNA expression of AKT\ERK1 in each group was detected by RT-PCR.The results showed that compared with NC group,the expression of AKT\ERK1 gene in M group increased,and the expression of AKT\ERK1 gene in I group decreased（P<0.05）,suggesting that overexpression of miR-126 in BMSCs may promote AKT/ERK1 gene expression.4.The expression of AKT\ERK1 protein in each group was detected by western blot.The results showed that the expression of AKT\ERK1 protein in M group was significantly increased and the expression in I group decreased compared with NC group（P<0.05）,suggesting that overexpression of miR-126 in BMSCs may promote AKT/ERK1 protein expression.5.The proliferation of RBMVECs co-cultured with BMSCs was detected by CCK-8assay.The results showed that compared with NC group,the proliferative ability of RBMVECs in M group increased and the proliferation of RBMVECs in I group decreased（P<0.05）.It suggested that under the condition of simulated intracerebral hemorrhage,miR-126 overexpressed BMSCs could enhance the proliferation of RBMVECs co-cultured with it.Conclusions:1.Under simulated cerebral hemorrhage in vitro model conditions,miR-126 overexpression can increase the expression of AKT\ERK1 in BMSCs.2.Under simulated cerebral hemorrhage in vitro model conditions,BMSCs with miR-126 overexpressed could enhance the proliferation of co-cultured RBMVECs.