MiR-124 Target Gene Screening And MiR-124 Facilitates Neurite Outgrowth And Elongation By Down-regulating The OSBP Expression

microRNA,a class of endogenous 22 nucleotide noncoding RNA,repressed gene expression by imperfectly pairing with nucleotide sequences within the 3'-untranslated region of targets,making them became central regulator of gene regulation.It was now evident that hundreds of miRNA genes had the power to regulate the expression of at least one-third of the human genome.The CNS was a rich source of miRNA expression system and the diversity of miRNA functions affected a fairly large number of neuronal genes.miRNA were related to in almost all biological processes,such as differentiation,death,apoptosis,inflammation and also their expression was highly regulated by specific enzymes or by epigenetic mechanisms.Aberrant expression patterns of microRNA resulted in a wide range of diseases,including cancers,inflammatory and developmental disorders.When transfected into non-neuronal HeLa cells and induced a neuronal gene profile,the miR-124 was first to be known.During the embryonic CNS development,the miR-124 suppressed the expression of SCP1 to induce neurogenesis.miR-124 promoted nervous system development by down-regulating the expression of PTBP1 and triggering a downstream switch to global nervous system-specific alternative splicing.Several studies found miR-124 acted as a neuronal fate determinant in the subventricular zone and played a role in adult neurogenesis.Moreover,recent studies found that miR-124 had been observed in stimulating the neurite outgrowth and elongation during neuronal differentiation and also suggested miR-124 could suppress ROCKI to promote neurite outgrowth.But the neurite outgrowth process was complicated.So the exact target genes remained completely unknown.The Oxysterol-Binding Protein(OSBP),located on the human chromosome 11 and mouse chromosome 19,was first identified and purified from Hamster Liver Cytosol in the 1980s.OSBP belonged to the oxysterol-binding family that was composed of 11 other OSBP-related proteins(ORPs).This family was closely related to regulate lipid and sterol homeostasis,vesicle transfer and cell signaling and also regarded as a Sterol and phosphoinositide sensors mediator of the oxysterol feedback regulation of cellular cholesterol homeostasis.However,the role that the OSBP played in the central nervous system remained largely enigmatic.HDACs belonged to a class of enzymes that had the ability to remove acetyl groups from lysine amino acid on a histone or non-histone proteins.Mammalian HDACs were usually subdivided into three classes based on their similarity to yeast HDACs.Class I members(HDAC 1,2,3,8 and 11)were homologous to the yeast RPD3 protein.Class ? HDACs(HDAC4,5,6,7,9 and 10)had similarities to yeast HDA1.The third class was the sirtuin proteins.They were homologous to the yeast SIR2 protein.Class ? histone deacetylases(HDACs)had a weak active deacetylase domain,but had a long N-terminal domain to bind transcription factors.It was previously reported that HDAC4 was regulated by miRNA such as miR-129a,miR-365 and so on.There were miR-124 target sites in the HDAC4 3'UTR,However whether HDAC4 directly regulation by miR-124 remained unknown.Recent studies found that miR-124 had been observed in stimulating the neurite outgrowth and elongation during neuronal differentiation but the exact target genes were remain completely unknown.So the destination of this paper was to explore the direct target of miR-124.At first We used the online microRNA prediction program TargetScan to investigate its potential target and found four conserved miR-124 target sites in the 3'UTR of OSBP.To confirm whether OSBP was the direct target of miR-124,the OSBP 3'UTR was cloned into a dual-luciferase reporter vector(psiCHECKTM-2 Vector).HEK293 cells were co-transfected with the resulting construct and hU6-hsa-miR-124-CMV-GFP(U6-124)plasmid or the two control plasmids hU6-scrambled-CMV-GFP(U6-124mut)and hU6-hsa-CMV-GFP(U6).Dual-Luciferase reporter assays showed a significantly lower expression(almost 50%reduction)of the luciferase gene in the presence of miR-124,compared to the negative control.When the miR-124 target sequences in OSBP were mutated("GTGCCUU",the underlined nucleotides were mutated into "CGG"),the inhibitory effect of miR-124 was abolished completely.Having established the ability of miRNA124 to interact directly with the OSBP 3'UTR,We next examined its effect on endogenous expression of OSBP.HEK293 cells were transfected with miR-124 over-expression plasmid or its control plasmid.We observed a significant decrease of OSBP protein in the miR-124 over-expressing cells compare with the cells transfected with the control plasmid.Our data indicated that OSBP was a potential direct target of miR-124.We also used the same means to explore whether the HDAC4 was the direct target of miR-124.Four conserved miR-124 target sites were found in the 3'UTR of HDAC4.The Dual-Luciferase reporter assays showed a significantly lower expression of the luciferase gene in the presence of miR-124,compared to the negative control.When the miR-124 target sequences in HDAC4 were mutated,the inhibitory effect of miR-124 was abolished completely.We observed a significant decrease of HDAC4 protein in the miR-124 overexpressing cells compare with the cell transfected with the control plasmid in HEK293 cells.Our data indicated that HDAC4 was a potential direct target of miR-124.It was previously reported that miR-124 expression was up-regulated during mouse brain development,with peaks at E14 and E17 and highly repressed at postnatal.To study whether the expression patterns of OSBP and miR-124 was opposite,we used different methods to prove it.RT-PCR result indicated that the expression level of OSBP was significantly decreased during the brain development compared to the control embryonic day 18.A decrease in the protein level of OSBP was detected using Western blotting analysis in cerebral cortex tissue of different stage.The Immunofluorescence staining also showed that the expression of OSBP in cortex was decreased in 8 week old mice compared to the 4 week old mice.Our study indicated that the trend in OSBP expression was negatively correlated with miR-124 during brain development.We had known OSBP might be the direct target of miR-124 and it was reported that over-expression of miR-124 promoted neurite outgrowth.The next step we would study the function of OSBP in stimulating neurite outgrowth and elongation.To explore the function of OSBP in stimulating neurite outgrowth and elongation during the cell differentiation,two special short hairpin RNA(shRNAs)against OSBP were synthesized.To verify whether this two shRNAs could down-regulate the expression of OSBP,We transfected the shRNA and pLVX-IRES-tdTomato-OSBP into HEK293 cells.Both shRNA-1 and shRNA-2 remarkably reduced expression of OSBP at the protein levels.Then we examined whether knockdown of OSBP with RNA interference affected the neurite outgrowth in N2a cells.Neurite outgrowth assay indicated that shRNA-1 significantly facilitated the neurite growth and increased the numbers of two cell body diameters.Our study indicated that down-regulation of OSBP promoted neurite growth in N2a cells.It was reported that the over-expression of miR-124 promoted neurite outgrowth in P19 cells.We then investigated whether miR-124 had a similar effect on neurite elongation in N2a cells.The over-expression of miR-124 in the N2a cells increased neurite outgrowth and elongation,as shown by fluorescent microscopy analysis.Quantitative analyses showed 34.12±3%greater two cell body diameters than in controls as soon as 24 h after the RA-induced neural differentiation.Then we examined the consequence of the ectopically expressed OSBP in miR-124 stimulated neural diffrerntiation and Neurite Elongation,we contransfected N2a cells with hU6-has-miR-124-CMV-GFP and pmCherry-OSBP or empty pmCherryNl plasmid as a control.Result revealed that over-expression of OSBP inhibited the effects of miR-124 on neurite outgrowth.To further illustrate the role of OSBP in neurite growth,we transfected mouse cortical neurons with shOSBP2 or shcontrol as a control.The results showed that the neurons exhibit much longer neurite following the OSBP down-regulation using special shRNA2.In addition,there was a significant decrease in neurite outgrowth after transfected with pmCherry-OSBP plasmid than the cells transfected pmCherry-N1.These data suggest that silencing of OSBP gene by shRNA promoted neurite growth while over-expression OSBP suppressed neurite growth.Together,these data suggested that OSBP played a critical role in the neurite outgrowth induced by miR-124.We also checked the function of HDAC4 in stimulating neurite outgrowth and elongation.miR-124 over-expression plasmid U6-124 was co-transfected with HDAC4-Tomato or his control plasmin td-Tomato into N2a cells.The results found that there was no difference in neurite outgrowth between the experimental group and the control group.These results suggested that miR-124 induced neurite outgrowth was not abolished by HDAC4 over-expression in N2a cells.In conclusion,we demonstrated,for the first time,that HDAC4 and OSBP might be the direct target of miR-124.In N2a cells,miR-124 promoted the neurite outgrowth by targeting OSBP.However miR-124 induced neurite outgrowth was not abolished by HDAC4 over-expression.In N2a cells and mouse cortical neurons,silencing of OSBP gene by shRNA promoted neurite growth while over-expression OSBP suppressed the neurite growth in mouse cortical neurons.