| The traumatic spinal cord injury (SCI) due to auto and sports related toaccidents, which often occurs at young and working age, is a devastatingexperience with a poor prognosis affecting mobility and early mortality.Recently, the trend of incidence in SCI has a noticeably increasing in ourcountry. There are more than60thousands patients suffering from this diseaseevery year. However, there are still no effective ways to cure SCI in an idealfunctional recovery. Over the years, many people, including scientists andclinical doctors, did believe that the nerves in spinal cord lost their capability toregenerate after injury. But recent researches showed that the nerve regenerationcould occur after SCI if the environment at lesion site was good enough.Currently, the key molecules and mechanisms of SCI are still unclear. Therefore,it is very important to explore the new molecules and mechanisms closelyrelated to injury and repair for treatment of SCI in future.MicroRNAs (miRNAs) are a newly discovered class of small noncoding RNAs including about22nucleotides. They negatively regulate gene expressionat posttranscriptional level. It is predicted that the~1000miRNAs in the humanbody may regulate the expression of up to1/3of human protein-coding genes,which implies the potential influence of miRNAs on almost every geneticpathway. Existing researches show that many miRNAs have extensiveexpression in the central nervous system and play important roles indevelopment and function. MiRNAs have been evaluated for their roles in brainischemia, traumatic brain injury and spinal cord injury. In addition, microRNAsare implicated in neurodegenerative disorders, including Alzheimer s disease,Huntington disease and Parkinson disease.After detected the expression of miRNA from normal and injured spinalcord tissues at1,3,7,14,21d, and28day postinjury (dpi) of C57BL/6mice byAgilent Mouse miRNA array, eleven miRNAs were found down-regulated orup-regulated in spinal cord tissues from spinal cord injured mice. Theexpression of miR-21was up-regulated at3d,7d and14d. The expression ofmiR-142-3p and miR-223were up-regulated at3d and7d. The expression ofmiR-146a was up-regulated at14d,21d and28d. The expression of miR-199bwas up-regulated at7d. While the expression of miR-1was down-regulated at7d,14d,21d and28d. The expression of miR-133a and miR-133b weredown-regulated at21d and28d. The expression of miR-150and miR-486weredown-regulated at7d. Quantitative real time PCR confirmed the results.Unexpectedly, the expression of miR-124wasn t up-regulated ordown-regulated after SCI, which is one of the most conserved and abundantlyexpressed miRNAs in central nervous system. We presumed that the result wasconcerned with the length of the spinal cord we extracted. We stained thesections of brain and spinal cord by in-situ hybridization, and found that miR-124was abundantly expressed in olfactory bulbs, hippocampus, cerebralcortex, cerebellar peduncle and gray substance of spinal cord. We extracted thetotal RNAs from rostral and caudal0.8cm length spinal cord tissues of lesionsite. The expression of miR-124was detected by qRT-PCR. We found that theexpression of miR-124was significantly down-regulated at1,3and7dpicompared with the control mice. Then we performed double-staining within-situ hybridization in miR-124and immunohistochemistry in NeuN, a neuronspecific marker, detection respectively. The results showed that miR-124andNueN did not express in the epicenter of lesion stie at7dpi. At the pericenter oflesion site, we found that some NeuN-positive cells didn t express miR-124.While NeuN and miR-124were both expressed in the normal spinal cordneurons. These data indicated that the expression of miR-124was closelyrelated to spinal cord injury. MiR-124probably participated in the regulation ofspinal cord neuron plasticity and played roles in the process of SCI.The number of predicted target genes of miR-124are more than onethousand by bioinformatics retrieve, and the number of target genes relative tothe central nervous system was probably one hundred. Owing to the huge works,we couldn t validate the predicted target genes one by one by dual-luciferasereporter assay. There are three general methods for miRNA in loss-of-functionstudy: genetic knockout, antisense oligonucleotide inhibitors and miRNAsponges. Certainly, genetic knockout is the best way to research gene function.However, miR-124is encoded by three distinct genes locating at differentchromosomes. It is impossible to completely knockout miR-124encoded genesfrom genome. While antisense oligonucleotide of miR-124performs a transientinhibition and isn’t suitable for the study of chronic diseases in vivo. So westudy the miR-124function by means of miRNA sponges which can knock down endogenous miRNA. Typical miRNA sponge constructs contain seven toten binding sites separated by a few nucleotides each which can inhibit miRNAfunction by complementation fixation with mature miRNA. Firstly, wesynthesized plasmid containing7binding sites which were perfectlycomplementary in the seed region with a bulge at the position9-12to preventRNA cleavage and degradation. Then we subcloned the binding sequence topEGFP-C3and pMIR-report vector. The constructed new plasmids namedpEGFP-miR-124sp and pMIR-miR-124sp. pEGFP-miR-124sp containedenhanced green fluorescent protein (EGFP) reporter gene and pMIR-miR-124sphad a luciferase reporter gene. We transfected sponges vectors and miR-124mimic into COS-7cells to validate the efficacy of miR-124sponges vector.When we cotransfected miR-124mimic and pMIR-miR-124sp to COS-7cells,the result showed that the activity of firefly luciferase was down-regulatednearly90%compared with the control group. Retrieve experiment confirmedthat the activity of firefly luciferase didn t change when we cotransfectedpEGFP-miR-124sp, pMIR-miR-124sp and miR-124mimic. When wecotransfected miR-124mimic and pEGFP-miR-124sp, the result showed that theexpression of EGFP was visibly down-regulated compared with the controlgroup under fluorescence microscope. Then we detected the EGFP expression intransfected cells by flow cytometry, and got the same results. These dataintensively indicated that miR-124sponges vector could knock down miR-124mimic in COS-7cells. So we successfully constructed the miR-124spongesvector.Next, we constructed the pCIG-miR-124vector that could highly expressedmiR-124precursor. When pCIG-miR-124and pCIG were transfected into Helacells, we could observe the expression of green fluorescent protein under the fluorescence microscope. The miR-124expression of Hela cells tranfectedpCIG-miR-124was significantly higher than that tranfected pCIG. The resultshowed that pCIG-miR-124vector could express mature miR-124in Hela cells.The miR-124highand miR-124lowtransgenic mouse model were established bypronuclear injection of fertilized eggs. And we obtained nine EGFP positivemiR-124lowtransgenic mice and six EGFP positive miR-124hightransgenic mice.Furthermore, we observed weak fluorescence signals in miR-124lowtransgenicmice spinal cord and strong fluorescence signals in miR-124hightransgenic micespinal cord. The result preliminarily indicated that miR-124highand miR-124lowtransgenic mice were successfully established. We hope that we can elucidatethe regulation mechanisms of miR-124in the process of spinal cord injury basedon the study of miR-124highand miR-124lowtransgenic mice. |
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