Regulation Of MiR-184 On Angiogenesis After Acute Cerebral Infarction And Its Mechanism

Objective:Cerebrovascular disease(CVD)is a severe threat to human health because of its high disability and mortality.Ischemic stroke(IS)accounts for 60%-80% of stroke.Ischemia leads to degeneration and necrosis of most nerve cells.The formation of neovascularization in the ischemic area is conducive to collateral circulation,improving blood supply capacity,and recovery of nerve function,It is the primary method of IS treatment.Previous studies found that the expression of mi R-184 was decreased in ischemic brain tissue.Through target gene prediction analysis,it was found that the 3'UTR of phosphatidyl acid phosphatase 2B(PPAP2B)contained a nucleotide sequence paired with mi R-184.Therefore,we speculate that mi R-184 may participate in angiogenesis and play a role in ischemic brain tissue through the target gene PPAP2 B.In this study,we observed mi R-184 and PPAP2 B expression after cerebral ischemia and hypoxia in vivo.We followed the effect of overexpression and inhibition of mi R-184 on angiogenesis of endothelial cells and the changes of PPAP2 B vascular endothelial growth factor(VEGF)signal in vitro,To explore the regulatory effect of mi R-184 on angiogenesis of ischemic stroke and clarify its mechanism,to provide a new strategy for clinical treatment of IS and improve the prognosis of patients with IS.Methods:Our study mainly includes five parts.The first part is the study of micro RNA expression profile in patients with acute cerebral infarction(AIS): the changes of serum mi RNAs in patients with AIS were analyzed by sequencing.The specific mi RNAs in patients with AIS were screened.QRT-PCR verified the mi RNAs with apparent differences,and the target genes were analyzed and screened.In the second part,we established the SD(Sprague Dawley)rat model of middle cerebral artery occlusion/reperfusion(MCAO/R),We injected the Ad-mi R-184 vector and ad NC vector into the right lateral ventricle to explore the neuroprotective effect of mi R-184after cerebral ischemia,TTC staining was used to observe the volume of cerebral infarction,MNSS score was used to evaluate the neurological deficit,and QRT-PCR and Western blot were used to detect the m RNA and protein expression of PPAP2B and VEGF.In the third part,the oxygen and glucose deprivation/reperfusion(OGD/R)cell model was established to detect the expression of mi R-184 and its effect on cell proliferation,apoptosis,and angiogenesis.q RT-PCR and Western blot detected the m RNA and protein expressions of PPAP2B and VEGF.In the fourth part,we transfected mi R-184overexpression lentivirus(OE-mi R-184)and mi R-184 silencing lentivirus(SP-mi R-184).By adjusting the expression of mi R-184,we observed the effects on cell proliferation,apoptosis,angiogenesis,and the changes of PPAP2B and VEGF m RNA and protein levels.In the fifth part,the target gene PPAP2B was interfered with by lentiviral vector interference technology to observe whether the regulation of mi R-184 on angiogenesis depends on PPAP2B.Finally,we verified the binding target of mi R-184 and PPAP2B though a double-luciferase experiment.Results:(1)A total of 503.74 m clean reads were obtained from 25 samples.Each sample was no less than 13.48 m clean reads.Five thousand eighty-four mi RNAs were detected,including 2007 known mi RNAs,3077 new predicted mi RNAs,and differentially expressed 236 mi RNAs,which includs153 up-regulated mi RNAs and 83down-regulated mi RNAs;the mi RNA expression abundance of each sample was quantified to obtain the screening results of differentially expressed mi RNAs;19815mi RNA target genes were predicted,and the functional annotation and enrichment analysis of differentially expressed mi RNA target genes were completed.(2)The middle cerebral artery ischemia(MCAO)in SD rats was successfully established.The expression level of VEGF in the MCAO/R group was higher than that in the sham operation group on the first day after MCAO/R,suggesting that cerebral ischemia injury can lead to reactive angiogenesis.The results of real-time quantitative PCR showed that the expression level of mi R-184 in the ischemic cerebral cortex after MCAO/R was lower than that in the sham operation group,and the expression level of PPAP2B m RNA and VEGF in the MCAO/R group was higher than that in the sham operation group.Western blot results show that PPAP2B and VEGF protein expression in the ischemic cerebral cortex increased after MCAO/R compared with the sham operation group.(3)The expression of mi R-184 in SH-SY5Y cells decreased after 24 hours of glucose and oxygen deprivation reperfusion,while the proliferation,and angiogenesis of SH-SY5Y cells were enhanced.At the same time,the expression of PPAP2B and VEGF in m RNA and protein levels also increased,suggesting that mi R-184 has an inhibitory effect on angiogenesis.Overexpression of mi R-184 inhibited the lumen formation of vascular endothelial cells,At the same time,inhibition of mi R-184 promoted the lumen formation of vascular endothelial cells and significantly increased the m RNA and protein levels of PPAP2B and VEGF.After PPAP2B interference lentivirus down-regulated PPAP2B and VEGF levels,mi R-184 reversed the ability of lumen formation and proliferation of vascular endothelial cells.Conclusions:The expression of mi R-184 decreased after acute cerebral infarction.Mi R-184 has a neuroprotective effect on MCAO rats.And mi R-184 can regulate angiogenesis after oxygen and glucose deprivation(OGD).This regulatory effect may depend on the inhibition of PPAP2 B by mi R-184.Mi R-184 may become a therapeutic target for acute cerebral infarction.