| Schizophrenia is a chronic and severe mental illness characterized by a range of cooccurring symptoms,including hallucinations,delusions,affective flattening,social withdrawal,cognitive impairment,etc.The pathogenesis of schizophrenia are not well understood and treatment options are limited,thus it is profoundly significant to explore the underlying mechanisms of schizophrenia and find its therapeutic targets.MicroRNAs(miRNAs)are non-coding RNAs that interact with the 3’UTR of the target mRNA through sequence-specific base pairing to degrade the mRNA or inhibit translation,resulting in downregulating(silencing)the expression of the target gene.Each miRNA may regulate the expression of hundreds of target genes,thus the dysfunction of miRNAs,especially when the dysfunction occurs in the central nervous system,is of great clinical significance and poses a major challenge to both investigate the underlying molecular mechanisms of diseases and develop effective therapeutic approaches.There is ample evidence that miRNAs play an important role in the development of schizophrenia.MiR-144/451 is a double cis-trans locus encoding miR-144 and miR-451.A large amount of data suggests that miR-144/451 is involved in the regulation of various neuropsychiatric disorders such as depression,autism,Alzheimer’s disease,Parkinson’s disease,etc.,however,whether it has a regulatory role in schizophrenia has not been studied.Based on the above background,this study aims to clarify whether miR-144/451 has a regulatory role in schizophrenia and to elucidate the possible molecular mechanisms to provide a new miRNA target for schizophrenia.This study was divided into following 3 parts:Part Ⅰ:Modulatory effect of miR-144/451 knockout on phenotypes of schizophrenia mouse modelObjective:To obtain wild-type(WT)mice and miR-144/451 knockout(KO)mice from the same litter by genetic breeding and establish the schizophrenia mouse model to determine whether miR-144/451 knockout has a modulatory effect on the schizophrenia phenotypes.Methods:1.WT and KO mice from the same litter were randomly divided into conWT group,SZWT group,conKO group and SZKO group respectively.The mouse model of schizophrenia was established by continuous intraperitoneal injection of(+)-MK-801 for 14 days at a dose of 0.2mg/kg,with an equal amount of saline as control.2.To evaluate the symptoms of schizophrenia in mice:the degree of spontaneous activity(positive symptoms)in mice was detected using the open field test(OFT),the state of sensory gating function in mice was detected by the prepulse inhibition of the startle reflex(PPI),the cognitive function was measured by the nesting test and the degree of anxiety and depression(negative symptoms)was detected by the forced swimming test(FST).3.The mRNA levels of miR-144 and miR-451 in the hippocampus of conWT and SZWT groups were detected by in situ hybridization.4.Enzyme-linked immunosorbent assay(ELISA)was applied to detect prefrontal cortical and striatal dopamine levels in mice.Results:1.In the OFT,there was no significant difference in the distance travelled and average speed of locomotion between conWT and conKO mice(both P>0.05),and intraperitoneal injection of MK-801 significantly increased the distance travelled and average speed of SZWT and SZKO mice,i.e.,the mice were spontaneously hyperactive(all P<0.05)while the spontaneous activity of SZKO mice was significantly reduced compared to SZWT mice(P<0.05).In the PPI,PPI%of conKO mice was significantly decreased compared with conWT mice(all P<0.05).MK-801 injection significantly decreased PPI%of SZWT mice(all P<0.01)while significantly increased PPI%in SZKO mice at 80 dB and 85 dB prepulse stimulation(both P>0.05).PPI%of SZKO mice was significantly higher than SZWT mice(all P<0.05).In the nesting test,there was no significant difference in nesting scores between conWT and conKO mice(P>0.05).MK-801 injection significantly decreased nesting scores in SZWT mice(P<0.001)and scores of SZKO mice significantly increased compared to SZWT mice(P<0.01).In the FST,there was no significant difference in immobility time between conWT and conKO mice(P>0.05).MK-801 injection significantly prolonged the immobility time in SZWT and SZKO mice(both P<0.001)while SZKO mice had a significantly shorter immobility time compared to SZWT mice(P<0.001).2.MK-801 injection significantly increased miR-144 and miR-451 mRNA levels in the hippocampus of SZWT mice(both P<0.001).3.There was no significant difference in dopamine content in prefrontal cortex and striatum between conWT and conKO mice(both P>0.05).MK-801 injection significantly decreased the content of dopamine in the prefrontal cortex and increased the content of dopamine in the striatum of SZWT and SZKO mice(all P<0.001).The decrease in prefrontal cortical dopamine levels and the increase in striatal dopamine levels in SZKO mice were both significantly weaker than in SZWT mice(all P<0.01).Conclusion:Schizophrenia symptoms and dopamine dysregulation were significantly improved in miR-144/451 knockout mice,suggesting a significant regulatory effect of miR144/451 on schizophrenia.It was also observed that intraperitoneal injection of MK-801 increased miR-144/451 expression in hippocampus region of SZWT mice.Part Ⅱ:The modulatory role of miR-144/451 overexpression in the brain on the phenotype of schizophrenia mouse modelObjective:To clarify the regulatory effects of intracerebral overexpression of miR144/451 on the phenotype of schizophrenia.Methods:1.WT mice were randomly divided into con group,ago group,SZ group and SZago group.Intracerebral overexpression of miR-144/451 was achieved by stereotactic injection of a mixture of miR-144-3p and miR-451a agomir into the brain,with equal amount of miR-NC as control.Schizophrenia symptoms were induced by continuous intraperitoneal injection of(+)-MK-801 for 14 days at a dose of 0.2 mg/kg,with an equivalent amount of saline as a control.2.The spontaneous activity(positive symptoms)of mice was detected using the OFT and the cognitive function of mice was detected using the nesting test.3.The prefrontal cortex and striatal dopamine content of mice were detected by ELISA.Results:1.There was no significant difference in the distance traveled and mean speed of movement between con group and ago group in the OFT(both P>0.05).The distance travelled and the mean speed of activity in the SZago group were significantly higher than those in the SZ group(P<0.05).There was no significant difference in nesting scores between con group and ago group(P>0.05).There was no significant difference between SZago group and SZ group(P>0.05).2.There were no significant difference in prefrontal cortical and striatal dopamine level between con group and ago group(both P>0.05).Prefrontal cortical dopamine level was significantly decreased in SZago group compared with SZ group(P<0.001)and striatal dopamine content was significantly increased in SZago group compared with SZ group(P<0.001).Conclusion:Overexpression of miR-144/451 in the brain aggravates schizophrenia symptoms and dopamine dysregulation,suggesting again that miR-144/451 is involved in regulating schizophrenia.Part Ⅲ:Modulation of oxidative stress and neuroinflammation in the brain of the schizophrenia mouse model mediated by miR-144/451Objective:To investigate whether miR-144/451 regulates oxidative stress and neuroinflammation in the brain of the schizophrenia mouse model using prefrontal cortex samples and frozen sections collected from the Part Ⅰ.Methods:1.Kits were applied to detect the content of malondialdehyde(MDA),reduced glutathione(GSH),superoxide dismutase(SOD)and adenosine triphosphate(ATP)in the prefrontal cortex of each group of mice.RT-PCR was further applied to measure the content of antioxidant enzymes including NAD(P)H:quinone oxidoreductase-1(NQO1),superoxide dismutase 1(Sodl),superoxide dismutase 2(Sod2),glutathione peroxidase(Gpx1),and catalase(Cat)in terms of relative mRNA expression.2.The protein expression of apoptosis-related proteins B-cell lymphoma-2(Bcl-2)and Bcl-2-associated death promoter recombinant protein(Bad)and microglia marker ion calcium-binding bridging molecule 1(Iba-1)were assayed by Western blotting.3.The in situ expression of Iba-1 and neuronal nuclear antigen(NeuN)in the prefrontal regions of each group was detected by immunofluorescence double-staining method.4.The number of apoptotic cells in the prefrontal area of each group was detected by Tunel method.Results:1.MK-801 injection significantly increased MDA content in the prefrontal cortex of SZWT and SZKO mice(both P<0.001)with a significant increase in SZWT mice compared with SZKO mice.The contents of GSH,SOD,ATP and the relative mRNA expression levels of NQO1,Sodl,Sod2,Gpxl and Cat in the prefrontal cortex were significantly downregulated in SZWT and SZKO mice(all P<0.01),and SZWT mice were significantly decreased compared with SZKO mice(all P<0.01).2.Injection of MK-801 significantly increased the expression of Iba-1 protein and the number of activated microglia in the prefrontal cortex of SZWT and SZKO mice(all P<0.001),while the expression of Iba-1 protein and the number of activated microglia in SZWT mice were significantly higher than those in SZKO mice(all P<0.001).3.Injection of MK-801 significantly decreased the expression of Bcl-2 protein and increased the expression of B ad protein(all P<0.001),and the number of Tunel-positive cells in the prefrontal cortex of SZWT and SZKO mice(all P<0.001).The expression of Bcl-2 protein was significantly decreased in SZWT mice compared with SZKO mice,while the expression of Bad protein and the number of Tunel-positive cells were significantly increased in SZWT mice compared with SZKO mice(all P<0.001).Conclusion:miR-144/451 deletion may inhibit the progression of schizophrenia and alleviate schizophrenia symptoms by exerting anti-oxidative stress,anti-apoptosis and inhibiting microglia activation. |
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