The Identification And Functional Analysis Of Novel Microrna In Focal Cerebral Ischemia Rats After Fastigial Nucleus Stimulation

Earlier studies showed that fastigial nucleus stimulation （FNS） reduces tissue damage resulting from focal cerebral ischemia. The mechanisms of neuroprotection induced by FNS are not entirely understood; however, important data have been presented in the past two decades. MicroRNAs （miRNAs） are a newly discovered group of non-coding small RNA molecules that negatively regulate target gene expression. Our previous study suggests that miRNA may serve as mediators of the brain’s response to FNS that leads to endogenous neuroprotection. This study was designed to identify and characteristic the novel miRNAs obtained after FNS.Part 1 The Identificaiton of Novel miRNA in Focal Cerebral Ischemic Rats after Fastigial Nucleus Stimulation[Objective] To explore the unkown miRNA in focal cerebral ischemic rats after fastigial nucleus stimulation.[Method] Strict criteria, including hairpin RNA structures and homology analysis were applied to define novel miRNA candidates. RT-PCR technology was employed to verify the expression of novel miRNA.[Results] Using computational analysis,781 novel miRNA candidates were predicted, we identify a new miRNA （PC-3p-3469₄06）, temporarily named rno-miR-676-1. The novel rno-miR-676-1 was preferentially expressed in muscle and brain tissue.[Conclusion] rno-miR-676-1 （PC-3p-3469₄06） is a novel miRNA, which can form tipical hairpin structures and exist homologous in mammal.Part 2 The Role of rno-miR-676-1 in Focal Cerebral Ischemic Rats after Fastigial Nucleus Stimulation[Objective] To investigate the regulatory mechanisms of rno-miR-676-1 in focal cerebral ischemia reperfusion injury of rats with stimulation of cerebellar fastigial nucleus.[Method] （1） Spragu-Dawley （SD） rats were randomly divided into 3 groups:sham operation group, I/R injury group and FNS group. The expression of rno-miR-676-1 was detected by real-time qPCR. （2） Functional annotation on the novel rno-miR-676-1 was carried out by in vivo observations. TTC staining was employed to measure ischemic lesion volumes ration; the apoptotic cells were detected by TUNEL assay. （3） The putative targets were predicted using Findtar software. The miR-target relationship was validated by dual-luciferase reporter assays.[Results] （1） Compared with the sham-operated and I/R-only injury groups, miR-676-1 expression was significantly increased after FNS. （2） In vivo observations indicate that rno-miR-676-1 agomir treatment decreased the infarct volume, cell death and improved neurological outcomes, while rno-miR-676-1 antagomir treatment increased the infarct volume, cell death and reduced neurological outcomes. （3） AUP1, CCNJ, PDPK1, BCL2L2 and HRAS were predicted as potential targets of rno-miR-676-1. Dual-luciferase reporter assays showed that they are not authentic targets of rno-miR-676-1.[Conclusion] rno-miR-676-1 was upregulated in rats with cerebral ischemia after FNS. rno-miR-676-1 over-expression improved neurological outcomes, whereas the reduction of rno-miR-676-1 exhibited the opposite effect.