Research On The Target Genes And Mechnisam Of MiR-124a In Promoting Neurite Outgrowth

The generation of a particular cell type requires a specific and precise spatiotemporal control of gene expression at different levels. Recently, it has been demonstrated that, in addition to transcriptional regulation, the posttranscriptional level is also crucial for regulating gene expression and that microRNAs (miRNAs) are important players in this process. miRNAs are endogenous, small RNA molecules with a length of 19-21 base pairs (bp). They are present in a variety of organisms, including plants, invertebrates, and vertebrates. Several lines of evidence have shown that miRNAs play key regulatory roles not only in normal cellular processes but also in different pathologies. miRNAs represent a precise and efficient manner of posttranscriptional regulation of gene expression because of their tissue specific distribution. Another feature of interest is that many miRNAs do not act as on-off switches, but rather fine-tune gene expression profiles. miRNA-mediated fine-tuning usually occurs in the absence of mRNA degradation.Like other tissues and cells, the nervous system and neural cell lines also express miRNAs, some of which are enriched or unique in the tissue and neural cells (e.g., miR-9, miR-124a, miR-125, miR-128, and miR-129). The number of miRNA genes found to be expressed in the nervous system seems to be larger than that in many other organs, perhaps partly reflecting the fact that the nervous system contains many types and subtypes of cells. Toward understanding the complexity of miRNA expression, these studies have further revealed that anatomically distinct areas of the adult central nervous system (e.g., cerebellum, hypothalamus, and hippocampus) express similar miRNAs, but relative miRNA levels can vary significantly in different regions. miRNA expression during neuronal differentiation and neurodevelopment has also been investigated. When treated with all-trans-retinoic acid, embryonal carcinoma cells will terminally differentiate into neuron-like cells. Accompanied with the morphological changes, expression of miRNAs such as miR-9, miR-124a, and miR-125 is significantly induced over time, suggesting that these’ miRNAs may play a role in differentiation or cell fate determination, in addition to their potential functions in adults.miR-124a is completely conserved at the nucleotide level from worms to humans and is estimated to be the most abundant miRNA in the brain, accounting for 25-48% of all brain miRNAs. In addition, the human miR-124a-1 locus is located in the chromosome 8p23 region, which is rich in genes that have been implicated in neuropsychiatric disorders, microcephaly and epilepsy. Overexpression of miR-124a in HeLa cells leads to the suppression of a large number of non-neuronal transcripts. Moreover, a neurogenesis suppressor gene, Ctdspl, and a neuron-specific splicing repressor gene, Ptbpl, have been identified as miR-124a target genes in vitro, and an increase of Ptbpl mRNA was observed in the telencephalon of a Dicer conditional knockout mouse. In vivo knockdown of miR-124a in mouse SVZ cells identified Sox9, a neurogenesis suppressor gene, as a miR-124a target, suggesting that miR-124a controls neurogenesis through suppression of Sox9 translation. One study found that miR-124a is required for neuronal determination in the developing chick neural tube. On the other hand, another study reported that miR-124a is not involved in the initial neuronal differentiation in the developing chick spinal cord. Dicer conditional knockout mice exhibited initial neurogenesis in the absence of miRNA production. Considering these inconsistent observations, the target genes of miR-124a and its functional role in neural differentiation remain ambiguous.A functional role for miRNAs in more specific neurological processes is also emerging, and their dysfunction could have direct relevance for our understanding of neurodegenerative disorders. Since miR-124a is expressed abundantly in differentiating and differentiated neurons and may target hundreds of mRNAs, it is possible that miR-124a may affect many aspects of the neuronal differentiation process such as neurite outgrowth and synapse formation. Recent study has found miR-124a is implicated in the process of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton. But the specific geires and mechanism that contribute to this process are still rarely known.Previous studies mostly explore the functional role of miRNA in the way by overexpressing or inhibiting the expression of a specific miRNA in relevant cell lines by using artificial synthetic miRNA mimics or miRNA inhibitors. A complete set of mRNA transcripts (the transcriptome) is prepared from the cells of an experimental treatment as well as a control library constructed from an untreated source. Then the cDNA microarray assay technology was preformed and the differentially expressed genes can be further studied. The obvious advantages of this method is that massive messages can be obtained in a single experiment. This facilitate the further study of a few differentially expressed genes on the one hand and the integrated analysis of the functional roles of the miRNA using the Bioinformatics Method on the other hand. However, the cDNA microarray method also has many disadvantages. For one thing, the activity of life in an organism is a multi-level stereo complex network. Many points are interconnected and one point may influence many other points. Therefore it is very difficult to discriminate which genes are the direct targets of a miRNA. For another thing, recent studies have found there are two major mechanisms by which a miRNA regulates the expression of a gene; one is mRNA degradation, another is translation inhibition. mRNAs regulated by the former mechanism can be readily detected by the microarray technique, but the information of the genes regulated by the later mechanism was always left out. For these reasons, we took advantage of the online bioinformatical tools combining with the candidate gene strategy to select the related genes that implicated in the early stage of nervous system development and neurite outgrowth. Then we test these selected genes by using the molecular biology technique. Moreover, we further study the roles and the mechanisms of these genes in the process of neurite outgrowth at cellular level.Chapter 1:The prediction and confirm of miR-124a target genesObjectivesGet the possible target genes of miR-124a that might participate in neurite outgrowth by taking advantage of the online bioinformatics service software TargetScan. Then construct the repoter plasmids containing the 3’untraslated regions (UTRs) of the relevant genes and test the role of these binding sites by transfecting of the reporter plasmids with the miR-124a overexpression plasmid in HEK293 cells.Methods1. Prediction and selection of miR-124a target genesBy using the widly used famous online miRNA target gene prediction software TargetScan, combining with the messages of the relevant genes in the literature, we selected DNMT3b gene, CAPN6 gene, HDAC4 gene, STAT3 gene, GSK3β, ROCK1, CBX2 gene and PPP1R13L gene as the candidate genes.2. Reporter plasmids construction of the candidate genesFirst of all, the the putative binding sites for miR-124a in the 3’UTR of the predicted genes were chosen. Next, primers with restriction enzyme sites according to the mRNA information in the Genbank were designed. The sequences amplificated should contain all the miR-124a binding sites predicted by the software. Then polymerase chain reaction (PCR) was performed to get the target sequences. The purified PCR products were ligated to the pMIR-REPORT plasmid or pGL3-Control plasmid to construct the reporter plasmids pGL3-Control-3’-ROCK1, pGL3-Control-3’-STAT3, pGL3-Control-3’-GSK3β,pGL3-Control-3’-CBX2, pMIR-3’-DNMT3b, pMIR-3’-CAPN6, pMIR-3’-HDAC4 and pMIR-3’-PPPlR13L. The plasmids were transformed to the E.coli DH5a cells. Clones were picked and macrocultured. The plasmids were extracted and the DNA fragments were sequenced to confirm the correction of the insert.3. Construction of the reporter plasmids containing the mutant miR-124a binding sitesWe designed a pair of point mutation primers. Using the 3’UTR plasmids of the candidate genes as template, we carried out point mutation PCR reaction with the High-Fidelity PCR Cloning Enzyme KOD-Plus-Neo DNA polymerase. Next the PCR product was digested by restriction endonuclease Dpnl to remove the methylated template. Then the DNA product was purified and transformed to the E.coli DH5a cells. Clones were picked and macrocultured. Then the plasmids were extracted and the DNA fragment was sequenced to confirm the correction of the insert.4. Luciferase reporter assayThe HEK293 cells were plated into the 48 well plates with 300 ul culture medium and without antibiotics. The reporter gene plasmids, the beta-galactosidase plasmid were cotransfected to the HEK293 cells with the miR-124a or miR-128 overexpression plasmids.24 hours after transfection the luciferase activity and the beta-galactosidase activity were assayed by using the Bright-Glo Luciferase Assay System from Promega and the High Sensitivity β-Galactosidase Assay Kit from Agilent corporation respectively. The β-Galactosidase plasmid was used to normalize the transfection efficiency.5. Western blot analysisWe transfected the human neuroblastoma cells BE(2)-M17 with the miR-124a expression plasmid.48 hours after transfection the cells were lysed by RIPA buffer and analysed by Western blot after determination of the protein concentration to see the protein level changes of ROCK1, CBX2 and PPP1R13L.Results:1.We chose the DNMT3b gene, CAPN6 gene, HDAC4 gene, STAT3 gene, GSK3β gene, ROCK1 gene, CBX2 gene and PPP1R13L gene as the candidate genes through bioinformatics analysis and referring to the relevant literature.2.3’UTR reporter plasmids pGL3-Control-3’-ROCK1, pGL3-Control-3’-STAT3, pGL3-Control-3’-GSK3β,pGL3-Control-3’-CBX2, pMIR-3’-PPP1R13L, pMIR-3’-DNMT3b, pMIR-3’-CAPN6 and pMIR-3’-HDAC4 were obtained.3.3’UTR point mutation reporter plasmids pGL3-Control-3’-mut-ROCK1 and pGL3-Control-3’-mut-CBX2 were obtained.4. Luciferase reporter assay indicated that miR-124a signifcantly reduced the luciferase activity of the pGL3-Control-3’-ROCK1, pGL3-Control-3’-CBX2 and pMIR-3’-PPP1R13L plasmid, but had no effect on the luciferase activity of the mutated 3’UTR reporter plasmids. miR-124a also had no effect on the luciferase activity of pGL3-Control-3’-STAT3, pGL3-Control-3’-GSK3β, pMIR-3’-DNMT3b, pMIR-3’-CAPN6 and pMIR-3’-HDAC4 reporter plasmids.5. Western blot analysis shown that the protein level of ROCK1, CBX2 and PPP1R13L in the miR-124a overexpression group was much lower than that of control group.Conclusions:We chose DNMT3b gene, CAPN6 gene, HDAC4 gene, STAT3 gene, GSK3β gene, ROCK1 gene, CBX2 gene and PPP1R13L as the miR-124a target genes and we found that miR-124a represses ROCK1 gene, CBX2 gene, PPP1R13L gene expression by interacting with the miR-124a binding sites in the 3’UTR of these genes.Chapter 2:Studying the role of ROCK1, CBX2 and PPP1R13L in neurite outgrowth Objectives:To explore the role of ROCK1, CBX2 and PPP1R13L in neurite outgrowth and elongation by using retinoic acid and miR-124a induced differentiation of M17 as cell model.Methods:1. Plasmid construction(1) Expression plasmids constructionTotal RNA was extracted from human neuroblastoma cell BE (2)-M17 and were reverse transcribed to complementary DNA (cDNA). We designed primers with restriction enzyme sites to amplify the coding sequences of these three genes by referring to the mRNA information in the GenBank. The PCR product was then purified and inserted into PmCherryNl red fluorescent protein fusion expression vector to construct ROCK1-Cherry, CBX2-Cherry and PPP1R13L-Cherry plasmids(2) Construction of the shRNA interference plasmidsThe interference sequences of these candidate genes were chosen by referring to the efficient sequences in the literature. The shRNA overexpression plasmids pGPU6-shROCKl-CMV-GFP, pGPU6-shCBX2-CMV-GFP and pGPU6-shPPP1R13L-CMV-GFP were constructed..2. Neurite outgrowth assay(1) miR-124a overexpression experimentThe human miR-124a overexpression plasmid hU6-hsa-miR-124a-CMV-GFP or the control plasmid hU6-CMV-GFP was transfected to the M17 cells by using Lipofectamine 2000. The cells were digested with trypsin 24 hours after transfection and re-plated on the poly-lysine coated 24 well cell culture plates. Retinoic acid(10uM)was added as soon as the cell attached to the plates to induce cell differentiation. The cells were observed 24 hours later by using fluorescence microscope and photos were taken.(2) RNA interference experimentThe shRNA expression plasmids for ROCK1, CBX2 and PPP1R13L were transfected to the M17 cells by using Lipofectamine 2000. The cells were digested with trypsin 24 hours after transfection and re-plated on the poly-lysine coated 24 well cell culture plates. Retinoic acid(10uM)was added as soon as the cell attached to the plates to induce cell differentiation. The cells were observed 96 hours later by using fluorescence microscope and photos were taken.(3) Gene overexpression experimentThe expression plasmids ROCK1-Cherry, CBX2-Cherry and PPP1R13L-Cherry were co-transfected to the M17 cells with the miR-124a expression plasmid hU6-hsa-miR-124a-CMV-GFP or the control plasmid hU6-CMV-GFP to the M17 cells by using Lipofectamine 2000. The cells were digested 24 hours after transfection and re-plated on the poly-lysine coated 24 well cell culture plates. Retinoic acid(10uM)was added as soon as the cell attached to the plates to induce cell differentiation. The cells were observed 24 hours later by using fluorescence microscope.3.Western blot methodP19 cells were induced to differentiate by retinoic acid (1 uM) and Western blot was performed to test the protein level change of the candidate genes during P19 cell differentiation.Results1. The coding sequences of ROCK1, CBX2 and PPP1R13L were confirmed by DNA sequencing to be inserted into the PmCherryNl plasmid.2. microRNA overexpression experiment revealed, when treated with retinoic acid(10uM), that hU6-hsa-miR-124a-CMV-GFP transfected M17 cells exhibited long neurites as rapidly as 24 hours, while the hU6-CMV-GFP plasmid transfected M17 cells did not show evident neurites.3. RNA interference experiment revealed, compared with the control plasmid pGPU6- CMV- GFP, knocking down any one of the three genes by transfection of plasmid pGPU6-shROCKl-CMV-GFP, or pGPU6-shCBX2-CMV-GFP or pGPU6-shPPP1R13L-CMV-GFP significantly promoted neurite extension in M17 cells 4 days after retinoic acid(10uM)induction.4.The effect of miR-124a on the neurite elongation was greatly inhibited when any one of the three candidate genes was overexpressed。5. The protein level of ROCK1, CBX2 and PPP1R13L decreased during the course of P19 cell differentiation.Conclusions:1. miR-124a can significantly increase the neurite length of differentiated M17 cells. This indicates miR-124a has a role in the early stage of neural differentiation.2. Knocking down any one of the three candidate genes promoted neurite elongation while overexpresson any one of the three candidate genes inhibited neurite elongation during the course of retinoic acid induced differentiation of M17 cells. These demonstrate miR-124a promotes early stage neural differentiation through repressing the expression of multiple genes that have inhibitory roles in neurite growth.Chapter 3 The downstream mechanism of miR-124a in promoting neurite development.In order to further explore the downstream mechanism of miR-124a in neural development, we chose the tested gene ROCK1 as the subject, referring to the previous literature and decided to study the possible relationship between ROCK1 and PI3K/AKT signal pathway.Methods:1. Drug treatment experiment(1) ROCK1 inhibitor Y-27632 (lOuM) was added to the culture medium of differentiated M17 or P19 cells. Cells were harvested at different time points and the changes of phosphorylated AK.T were tested by western blot.(2) The ROCK1 inhibitor Y-27632 (lOuM) alone or together with PI3K/AKT pathway inhibitor LY294002 (30 uM) was add to the differentiated M17 or P19 cells. The cells were harvested 24 hours later.(3) The M17 cells transfected with miR-124a expression plasmid hU6-hsa-miR-124a-CMV-GFP were subjected to retinoic acid (lOuM) induced differentiation. Then, the PI3K/AKT pathway inhibitor LY294002 (30uM) or DMSO was added to the cell culture medium. The neurite outgrowth discrepancy of different treatment groups was observed 24 hours later by using fluoresce microscope.2. Western blot:(1) The miR-124a expression plasmid or the ROCK1 shRNA plasmid was transfected to the M17 cells. Cells were harvested 48 hours later and the phosphorylated AKT was tested by western blot.(2) Cells were harvested at different time points during the course of P19 cell differentiation. Phosphorylated AKT were tested by western blot.Results1. When the ROCK1 inhibitor Y-27632(10uM) was added to the culture medium of differentiated M17 or P19 cells, the phosphorylated AKT increased.2. When M17 cells were transfected with the miR-124a expression plasmid or ROCK1 shRNA plasimid, the phosphorylated AKT increased 48 hours later.3. The ROCK1 inhibitor Y-27632 (10uM) significantly enhanced the neurite elongation of the differentiated M17 cells and P19 cells. This effect was greatly inhibited when the PI3K/AKT pathway inhibitor LY294002 (30uM) was added simultaneously.4. When the PI3K/AKT pathway inhibitor LY294002 (30uM) was added to the differentiated M17 cells transfected with miR-124a expression plasmid hU6-hsa-miR-124a-CMV-GFP, the neurite elongation stimulating effect of miR-124a was greatly reduced.5. During retinoic acid-induced differentiation of P19 cells, the protein level of ROCK1 decreased while the phosphorylated AKT increased.ConclusionsmiR-124a promoted neurite development partially by repressing the expression of ROCK1 gene. This leads to the activation of PI3K/AKT signal pathway. In consideration of the important role AKT played in neural development, it fully demonstrates the significant role miRNA-124a play in the posttranslational regulation of gene expression during neural development.