| Sleep deprivation(SD)refers to the lack of sleep quality or quantity due to various reasons.The 1/3 time of human life is spent in sleep,so the quality of sleep determines the quality of life left 2/3.With the rapid development of society,more and more people suffer from sleep loss,the World Health Organization reported that sleep deprivation has become a global problem affecting human health.Studies have shown that long-term sleep deprivation can cause a series of injuries to the body,for example,lack of sleep can increase the risk of cardiovascular disease,congestive heart failure and high blood pressure,causing immune dysfunction,increased infectious diseases.Secondly,sleep deprivation changed the body Endocrine and metabolic disorders,especially glucose metabolism,resulting in elevated blood sugar levels,increased insulin,and ultimately lead to insulin resistance,type 2 diabetes,obesity and hypertension.sleep deprivation is also an independent risk factor for stroke,lack of sleep increase the risk of stroke;Moreover,sleep deprivation damage the body's brain function,especially the hippocampus dependent learning and memory damage is currently recognized.Therefore,it is important to study the mechanism of sleep deprivation on tha hippocampus.Rodent sleep cycle is similar with human,therefore,the use of rat sleep deprivation model of sleep disorders,to solve the clinical problems to provide a theoretical basis.miRNAs is a class of non coding single stranded RNA molecules encoded by endogenous genes with length of about 22 nucleotides.The process of biogenesisi is complex.Firstly,The gene encode a hairpin structure of primary RNA(Pri-miRNA)via RNA polymerase II / III.Secondly,the RNase Drosha complex with DGCR8 cut into the precursor RNA(Pre-mi RNA).At last,intracellular nucleus Pre-miRNA transport to the cytoplasm,and is reprocessed by Dicer as a mature miRNA.The miRNAs is bound to the 3 'UTR region of target mRNA by base complementation,leads to the degradation of target mRNA or inhibition of protein synthesis,so as to achieve the negative regulation of target gene transcription.At present,more than 1600 kinds of miRNAs have been found in the human body,and more than 60% proteins have regulatory sites of miRNAs.Recently,the expression profile of miRNAs after sleep deprivation has been detected in vivo.The screening of miR-132 in the hippocampus of rats after sleep deprivation has been found by microarray technology.MiR-132 has the function of synaptic plasticity.Methyl CpG binding protein 2(MeCP2)as a related protein in Rett syndrome has been widely studied is caused by mutation of the MECP2 gene,a neurodegenerative disease.The lack of MeCP2 protein in hippocampal neurons can cause long-term potentiation(LTP)impaired.LTP was considered to be the molecular basis of learning and memory,and sleep deprivation can cause hippocampal dependent learning and memory impairment.Therefore,we hypothesized that sleep deprivation caused an upregulation of miR-132 in the hippocampus,which led to down-regulation of MeCP2 to affect hippocampal function.This study established rat modified multiple platform model of sleep deprivation to explore the expression of specific changes in the levels of miR-132 and MeCP2 in hippocampus after sleep deprivation,and to further explore the regulation of the MeCP2 and miR-132 after REM sleep of rats.Part? Effects of sleep deprivation on miRNAs in the hippocampus of rats Objective: To explore the effects of sleep deprivation on miR-132,miR-138,miR-127,miR-128 and let-7b in the hippocampus of rats.Method: SD rats were randomly assigned into five groups: control group,sleep deprivation 8h group(sd 8h),sleep deprivation 1d group(sd 1),sleep deprivation 3d group(sd 3),sleep deprivation 6d group(sd 6).The model of sleep deprivation was made by the modified multiple platform technique.The change of miR-132,miR-138,miR-127,miR-128 and let-7b in hippocampus were measured by RT-PCR via SYBRA green ?assay.Results: After REM slepp deprivation,the hair,weight and behavior of the rats are changed.As detected by RT-PCR:(1)In hippocampus,compared wirh control group,the level of miR-132 in sd 8h and sd 1 groups decreased significantly,while sd 3 group increased(P < 0.05).(2)There was a decrease in the level of hippocampal miR-138 in sd 8h and sd 3 groups as compared to control group,the level of miR-138 increased in sd 1 group(P < 0.01).(3)Compared with control group,the level of miR-127 in sd 8h,sd 1 and sd 3 groups was decreased,while increased in sd 6 group(P <0.05).(4)In hippocampus,the level of miR-128 in sd 8h group was higher than that of control group and was lower in sd 1 group than that in control group(P <0.05).(5)The level of let-7b increased in sd 8h,sd 1 and sd 3 groups as compared to control group.Conclusion: The changes of miR-132,miR-138,miR-127,miR-128 and let-7b in hippocampus were changed with the prolongation of sleep deprivation time.Part ? Effects of sleep deprivation on mecp2 in the hippocampus of rats Objective: To predict the regulation pathways of miR-132 in sleep deprivation based on the methods of bioinformatics and explore the expression of mecp2 in hippocampus after sleeping deprivation.Method: The UCSC genome browser,human miRNA disease database,Genecards database and TargetScan 6.2 were used to predict the downstream target genes of miR-132.Westrrn blot was used to detect the expression of mecp2 protein.Results: The UCSC genome browser shows that has-mi R-132 was located at 2049908~2050008 locus of human 17p13.3 chromosome and was 101 bp in length.It was conserved in several species.In human mi RNA disease database we found that miR-132 is associated with dementia,inflammation,supranuclear palsy,tumor and so on.Target Scan 6.2 shows that miR-132 may regulate multiple downstream genes associated with nervous system disorders,including mecp2.After REM sleep deprivation,in hippocampus the level of mecp2 in sd3 group was lower than control group,and the results between sd8 h,sd1,sd6 groups had no statistically significant difference.Conclusion: The bioinformatics analysis predicted the downstream target mecp2 gene of miR-132,and the expression of mecp2 protein was changes after REM sleep deprivation.Part ? The relationship between miR-132 and mecp2 in REM sleep deprivation Objective: To determine the relationship between miR-132 and mecp2 in REM sleep deprivation.Method: The sequences of mi R-132 and rno-mecp2-3'UTR were cloned and the reporter plasmids were constructed.The plasmids were identified by colony PCR.The relationship between mi R-132 and mecp2 was verified by double luciferase reporter assay.Results: PCR confirmed that the plasmid was successfully constructed.The double luciferase reporter gene suggested that miR-132 can't directly regulate the expression of mecp2 in REM sleep deprivation.Conclusion: miR-132 may indirectly regulate the expression of mecp2 in hippocampus of sleep deprivation. |
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