Study On The Role Of MiR-10b-5p In Cerebral Ischemia/Reperfusion Injury

Background and Objective: Ischemic stroke(IS)is a refractory disease caused by a thrombosis or an embolism in the artery supplying the brain which leads to the death of brain tissue and focal neurological deficits.Intravenous thrombolysis or mechanical thrombectomy to recanalize the occluded artery and to restore cerebral blood flow as soon as possible is the only effective therapy.Cerebral ischemia/ reperfusion injury(CIRI)is the most common complication of IS and a major causal factor of secondary cerebral injury.Up to date,there is no effective therapy available for CIRI.MicroRNAs(miRNAs)are a class of endogenous small noncoding RNA molecules involved in post-transcriptional regulation of gene expression.Abnormal changes in miRNA profile have been reported in the process of CIRI and miRNAs may serve as potential targets in developing new strategies for IS therapy.Basic fibroblast growth factor(bFGF)is a most studied neuroprotective agent for IS therapy.In our previous high-throughput miRNA sequencing study,the differential expression of miR-10b-5p within the ischemic penumbra of the rat insulted by CIRI was induced by exogenous bFGF treatment.No reports related with the study on the role of miR-10b-5p in CIRI have been found.Herein,the changes in the expression of miR-10b-5p and its predicted target mRNA induced by the treatment of exogenous bFGF to CIRI were studied in vivo using experimental CIRI animal model.The possible roles of miR-10b-5p in CIRI and their mechanisms were also studied using cell culture technique in vitro.Methods: 1.The target genes of miR-10b-5p were predicted by bioinformatics methods;2.The animal model of CIRI was constructed by occluding the left middle cerebral artery of a sprague dawley rat for 90 minutes.BFGF was administrated by intracerebroventricular injection following the reperfusion and the therapeutic effect of exogenous bFGF on CIRI was investigated;3.The influence of bFGF on the expression of miR-10b-5p and its predicted target gene in CIRI was examined by RT-qPCR;4.The expressions of miR-10b-5p and its predicted target gene in the PC12 cells transfected with miR-10b-5p agomir and miR-10b-5p antagomir were examined by methods of RT-qPCR and Western blot;5.The influence of bFGF on the expression of miR-10b-5p and its predicted target gene in PC12 cells insulted by OGD/R was examined by RT-qPCR;6.The effect of miR-10b-5p on PC12 cells proliferation was investigated by MTT experiment;7.The effects of miR-10b-5p on the viability and apoptosis of PC12 cells insulted by OGD/R were examined by MTT experiment and flow cytometry,respectively.Results: 1.17 genes are predicted to be the possible target genes of miR-10b-5p.Among the predicted target genes,LDHA is most closely related with CIRI and it might be the key target gene regulated by miR-10b-5p in the process of CIRI;2.The animal model of CIRI was successfully prepared and bFGF treatment could effectively protect the brain against CIRI demonstrated by improved neurological deficit.3.In the penumbra area and the corpus striatum of the brain with CIRI,the expression of miR-10b-5p was down regulated and the level of LDHA mRNA was up regulated by bFGF treatment.4.The results of RT-qPCR and Western blot indicatedthatoverexpression of miR-10b-5p could down regulate the expression of LDHA and miR-10b-5p inhibition could up regulate the expression of LDHA in PC12 cells;5.In the PC12 cells insulted by OGD/R,the expression of miR-10b-5p was down regulated and the level of LDHA mRNA was up regulated by bFGF treatment.6.The results of MTT experiment demonstrated that miR-10b-5p overexpression could inhibit the proliferation of low differentiated PC12 cells and miR-10b-5p inhibition could promote the proliferation of low differentiated PC12 cells.7.Overexpression of miR-10b-5p aggravated the cell damage caused by OGD/R,while inhibition of miR-10b-5p alleviated OGD/R induced cell damage.Conclusions: 1.The neuroprotection of exogenous bFGF on CIRI in rats and OGD/R in PC12 cells is associated with decreased expression of miR-10b-5p and increased expression of LDHA mRNA.2.MiR-10b-5p can negatively regulate the expression of LDHA and LDHA might be a key target gene of miR-10b-5p in CIRI.3.Overexpression of miR-10b-5p can aggravate CIRI and inhibition of miR-10b-5p can alleviate CIRI in rats.MiR-10b-5p might elicit its effect on CIRI through regulating the expression of LDHA,thereby affecting cell proliferation and apoptosis.