The Effects And Mechanism Of MiR-19a-3p On Glucose Metabolism And Apoptosis In Neurons After Ischemic Stroke

Objective: Stroke is one of the leading causes of death and disability worldwide,of which ischemic stroke accounts for about 80%.It places a huge burden on the patient’s family and society.However,the effects of ischemic stroke treatments are not satisfactory.Early thrombolytic therapy is currently recognized as the only effective treatment,but the time window is only 3～4.5h,which makes most patients lose the best opportunity.Micro RNAs(miRNAs)are endogenously expressed RNA molecules that inhibit m RNA translation and play important regulatory roles in the pathophysiological processes of various diseases.Among them,MiR-19a-3p is an important component of mir R-17-92 cluster.In recent years,it has been found to be associated with the pathogenesis of a variety of tumors,including lung cancer,gastric cancer,breast cancer,and hepatocellular carcinoma.In addition,studies have found that miR-19a-3p is overexpressed in glioma cells,and its expression level is closely related to the degree of malignancy of glioma.There are also reports that miR-19a-3p stimulates axon growth of embryonic cortical neurons.Furthermore,studies have confirmed that miR-17-92 clusters are overexpressed in cultured ischemic neural progenitor cells or in the subventricular zone of ischemic animals,significantly accelerating cell proliferation.At the same time,inhibition of the composition of the miR-17-92 cluster(miR-18 a and miR-19a-3p),cell proliferation is reduced,and cell death is enhanced.Therefore,we speculate that miR-19a-3p may play an important role in the pathogenesis of ischemic stroke,but there are few related studies.For this reason,we established a rat ischemia/reperfusion model and oxygen-glucose deprivation cultured neurons in vitro to study the functional role of miR-19a-3p in the pathogenesis of ischemic stroke and to explore its possible regulatory mechanisms in this study,to provide a theoretical basis for finding new targets for the treatment of the disease.Methods:1.The neurons and astrocytes in the hippocampus of newborn rats were isolated and cultured.Then,real-time quantitative polymerase chain reaction(q RT-PCR)was used to detect the difference in expression levels of miR-19a-3p in these two cells under normal conditions.Subsequently,oxygen-glucose deprivation(OGD)Neuronal model and cerebral ischemia-reperfusion(IR)rat model were established,,q RT-PCR was used to detect express changes of miR-19a-3p in neurons during ischemia and hypoxia in vitro and in vivo.2.The OGD and IR model were established.Western Blot was used to detect the levels of glucose metabolism-related enzymes in neurons after ischemic stroke.At the same time,the glucose uptake and lactic acid production of neurons were compared by colorimetry.TUNEL staining was used to detect the apoptosis of nerve cells,and the levels of apoptosis-related proteins were detected by Western Blot.Finally,transfecting the OGD model with miR-19a-3p mimic or miR-19a-3p inhibitor,the effects of miR-19a-3p on glucose metabolism and apoptosis in neurons after ischemic stroke were examined.3.Through bioinformatics analysis by Target Scan software,combined with the mechanism of glucose metabolism and ischemic stroke,we search for possible target genes of miR-19a-3p,and then use dual luciferase assay for targeted validation;q RT-PCR and Western Blot were used to detect the expression levels of m RNA and protein of target genes in OGD model;finally,siRNA of the target gene was transfected into neurons,either alone or along with the miR-19a-3p inhibitor,and glucose metabolism and apoptosis were evaluated,to verify the interaction of miR-19a-3p and its target gene.Results:1.Under normal conditions,the expression level of miR-19a-3p in astrocytes is higher than that in neurons;when ischemia and hypoxia,the expression level of miR-19a-3p is up-regulated in vitro or in vivo.2.After IR / OGD,the expression level of glucose metabolism-related enzymes decreased,the glucose uptake and lactic acid production of neurons decreased,the level of glucose metabolism decreased,the expression of apoptosis-related proteins increased,and the damaged neurons became apoptosis;In the OGD model,after the transfection of miR-19a-3p mimic,the level of glucose metabolism in nerve cells decreased,and the number of apoptotic cells increased,while the result of transfection of miR-19a-3p inhibitor was reversed.3.Bioinformatics analysis and double luciferase assay results suggested that ADIPOR2 might be a target gene for miR-19a-3p;under ischemia and hypoxia condition,the expression of ADIPOR2 m RNA and protein in nerve cells was reduced;These changes could be reversed by miR-19a-3p inhibitor,which improved the glucose metabolism and reduced apoptosis.Conclusions:1.The miR-19a-3p may play a regulatory role in the pathogenesis of ischemic stroke.2.The miR-19a-3p can reduce the glucose metabolism of nerve cells and increase cell apoptosis,indicating that miR-19a-3p can aggravate nerve damage after ischemic stroke.3.The miR-19a-3p may inhibit the neuronal damage of neurons in ischemic brain tissue after ischemic stroke by regulating ADIPOR2.The miR-19a-3p inhibitor can improve the glucose metabolism and reduce apoptosis of nerve cells during ischemia and hypoxia.It is expected to become a new therapeutic drug for ischemic stroke.