| Background:Parkinson's disease(PD)is a common neurodegenerative disease in the clinical The pathogenesis of PD was complex,specially for sporadic PD.The main pathological characteristics of PD are the loss of dopaminergic neurons in the dense part of substantia nigra and the accumulation of?-synuclein,resulting in the aggregation of lewy bodies,which was related to the mitochondrial dysfunction,autophagy disorders,oxidative stress damage and neuro-inflammation indicated bymany documents.miRNAs are endogenous non-coding single stranded RNA,which are highly conserved and modulate the expression of target genes at the transcription level.And the miRNA play important roles in almost all physiological processes of the organism,including growth and development,inflammation and apoptosis.Several lines of evidence demonstrated that a variety of miRNAs are abundantly expressed in the nervous system,and the abnormal expression are closely related to the occurrence and development of PD,for example,miR-7 and miR-135 are down-regulated in PD and regulate the the expression of?-synuclein directly.MiR-124 and miR-146a are highly expressed in the nervous system,and play important roles in neurological diseases such as glioma,encephalomyelitis and ischemic brain injury.However,the expression of miR-124 and miR-146a in Parkinson's disease and its mechanism with Parkinson's disease are still unclear.Part?The expression of miR-124 and miR-146a in Parkinson's disease mouse model and nerve cellsObjective:To detect the expression of miR-124 and miR-146a in brain tissue of MPTP-induced PD mice and in SH-SY5Y cells and BV2 cells induced in vitro.Methods:The mice were injected intraperitoneally with MPTP to establish the PD models.The relative expression of miR-124 and miR-146a in midbrain tissue at different time point were detected by qRT-PCR.In addition,the expression of miR-124 in dopaminergic neurons and miR-146a in microglia was detected by RNA in situ hybridization combined with tissue fluorescent immunostaining.QRT-PCR was used to detect the expression of miR-124 in the SH-SY5Y cells treated with MPP~+at different concentration and the expression of miR-146a in BV2cells treated with LPS at different concentrationsResults:Compared with the control,the expression of miR124 was significantly decreased,while the expression level of miR-146a was significantly increased in the MPTP model groups at the different time points after administration MPTP;And the midbrain dopaminergic neurons of MPTP model groups were significantly reduced,and the expression of miR-124 was weakened,while the expression of miR-146a was increased in the activated microglia;Compared with the control group,the expression of miR-124 were significantly decreased in SH-SY5Y cells treated with different concentrations of MPP~+(P<0.05),while the expression of miR-146a were increased in BV2 cells treated with different concentrations of LPS.Conclusions:The expression of miR-124 was decreased and the miR-146a was increased in the midbrain of MPTP induced mice models of PD and in the SH-SY5Y cells induced by MPP~+or BV2 cells treated with LPS,which suggested that the miR-124 and miR-146a might be biomarkers for early diagnosis of Parkinson's disease.Part?Prediction and identification of target genes of miR-124 and miR-146aObjective:To predict and verify the target genes of miR-124 and miR-146a.Methods:The target genes of miR-124 and miR-146a were predicted by the target gene prediction softwares:TargetScan,miRDB and Microrna.org and verified by dual luciferase report gene assay.SH-SY5Y cells were transfected with miR-124-mi or miR-124-NC,miR-146a-mi or miR-146a-NC,respectively,the mRNA and protein of target genes:Bim and IRAK1 were detected by qRT-PCR and Western blot.Results:miR-124-mi and miR-146a-mi could inhibit luciferase activity,Bim and IRAK1might a target gene of miR-124 and miR-146a,respectively.The expression of Bim mRNA and protein in miR-124-mi group were significantly lower than that in control group and miR-124-NC group,the expression of IRAK1 mRNA and protein in miR-146a-mi group were higher than that in control group and miR-146a-NC group.Conclusions:The Bim was a target gene of miR-124 and IRAK1 was a target gene of miR-146a.The expression of Bim and IRAK1 were regulated by miR-124 and miR-146a,respectively.Part ? The mechanism of miR124 and miR-146a participate in Parkinson's diseaseObjective:To explore the neuroprotective potential of miR-124 by regulating the expression of target gene Bim;and to investigate whether miR-146a participate in the neuroinflammation by regulating the target gene IRAK1.Methods:Stereotactic ventricle injection of miR-124 agonist or negative control,miR-146a inhibitor or negative control.MPTP treatment 2 days after administration were performed as previously:miR-124 agonist group,miR-124-NC group,miR-146a inhibitor group and miR-146a-NC group.The midbrain tissues of the mice in each group were collected 2 days after the last injection of MPTP.The expression of Bim and IRAK1 mRNA were detected by qRT-PCR,and Western blot was used to detected Bim,the apoptosis-related proteins:Puma,Noxa,Bid in cytoplasm and Bax in mitochondria,IRAK1 and NF-?B protein expression.ELISA was used to detect pro-inflammatory factors TNF-?,IL-1 and IL-6.TUNEL staining combined with immunohistochemistry was conducted to analyze the neuronal apoptosis.Results:Compared with normal mice,the expression of Bim mRNA and protein were increased in MPTP induced PD mice,and the expression of Bim mRNA and protein in the miR-124 agonist group was significantly lower than that in miR-124-NC group.In MPTP induced PD mice,the expression of Bid and mitochondrial Bax protein were up-regulated,but the expression of Bax in the miR-124 agonist group were significantly lower that in the miR-124-NC group.And there was no significant difference in the expression of Puma and Nox in each group.Compared with normal mice,the expression of IRAK1 mRNA and protein were decreased in MPTP induced PD mice,and the expression of IRAK1 in the miR-146a inhibitor group were higher than that of miR-146a-NC group.In MPTP induced PD mice,NF-?B was activated and the pro-inflammatory factors TNF-?,IL-1and IL-6were increased,but there was no significant difference in the levels of NF-?B,TNF-?,IL-1 and IL-6 between the miR-146a inhibitor group and the miR-146a-NC group.In MPTP induced PD mice,the apoptotic cells were significantly increased.The neuronal apoptosis rate in the miR-124 agonist group was significantly lower than that in miR-124-NC group.While there was no difference in neuronal apoptosis rate between the miR-146a inhibitor group and the miR-146a-NC group.Conclusions:In MPTP induced PD mice,miR-124 could reduce the neuronal apoptosisinduced by MPTP,by inhibiting the expression of Bim,reducing the downstream apoptosis protein Bax translocation into the mitochondria and inhibiting mitochondria mediated endogenous apoptosis;The study was the first to found that in MPTP induced PD mice,miR-146a inhibitor had little effect on the neuronal apoptosis induced by MPTP.miR-146a could inhibit the expression of IRAK1,but had no significant effect on the activation of NF-?B and the expression of TNF-?,IL-1 and IL-6,suggesting that miR-146a might be involved in neuroinflammation.Conclusion for full textThe study was the first to explore the expression of miR-146a in the brain tissues of Parkinson's disease,indicated that in the brain tissues of MPTP induced PD mice,the expression of miR-124 was down-regulated and the epression of miR-146a was up-regulated,which might be biomarkers for early diagnosis of PD.The experiment of stereotactic ventricle injection in vivo,found that the miR-124 played a neuroprotective role by inhibiting the expression of its target gene Bim,reducing the mitochondrial translocation of the downstream apoptosis-related protein Bax,and inhibiting mitochondria-mediated neuronal apoptosis.The role of miR-146a in PD was analyzed for the first time:miR-146a inhibited the expression of the target gene IRAK1 and involved in the regulation of neuroinflammation in the progress of PD. |
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