MiR-124a Promotes Neurite Outgrowth By Inhibiting The OSBP Expression

MicroRNAs(miRNAs)comprise a large family of 21-25-nucleotide-long RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans and plants,and have revolutionized our comprehension of the post-transcriptional regulation of gene expression.Recent reports show that most animal miRNAs imperfectly base-pair with sequences in the 3'untranslated region(3'UTR)of target mRNAs through seed sequence,and inhibit protein synthesis by either repressing translation or promoting mRNA deadenylation and decay.In mammals,miRNAs predicted to control the activity of approximately 50%of all protein-coding genes.Functional studies indicate that miRNAs participate in the regulation of almost every cellular process investigated so far and that changes in their expression are associated with many human pathologies.Studies have shown that the distribution of miRNA in organisms is tissue-specific.Like other tissues,the nervous system also expresses miRNA,some of which are enriched or unique in the nervous tissue and neural cells,such as miR-9,miR-124a,miR-125,miR-128,and miR-129.MiR-124a is completely conserved at the nucleotide level among species and expressed during the differentiation and maturation of neurons.Moreover,miR-124a is estimated to be the most abundant miRNA in the brain,accounting for 25?48%of all brain miRNAs.Most studies have shown the role of miR-124a in neurogenesis,but as the most abundant miRNA in nervous system,its role in neural development is still unclear.Recent researches have indicated that miR-124a may regulate the activities of the cytoskeleton and thus affect the formation of neurites.But how the miR-124a to regulate neurite outgrowth,and the direct target genes need to be further studied.Oxysterol-binding protein(OSBP)and OSBP-related proteins(ORPs)constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes.The mechanism of ORP proteins have remained incompletely understood so far.However,several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes,with impacts on signaling,vesicle transport,and lipid metabolism.The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling(OSBP),control of ER-late endosome(LE)contacts and LE motility(ORP1L),neutral lipid metabolism(ORP2),cell adhesion(ORP3),macrophage lipid homeostasis,migration and high-density lipoprotein metabolism(ORP8),apolipoprotein B-100 secretion(ORPIO),and adipogenesis(ORP11).An increasing number of protein interaction partners of ORPs have been identified,which involved in cell signaling,vesicle transport,lipid metabolism,glucose metabolism,regulation of cytoskeletal proteins,ion transport and so on,providing clues of their involvement in multiple aspects of cell regulation.Therefore,ORPs by binding to different ligands play the corresponding functions in organism.The expression level of ORPs family members exhibits tissue-specific.And,the ORP1 was transcribed at strikingly high levels in the cortical areas of human brain and displayed sterol-regulated expression in a cultured human neuroblastoma cell line.Thus,ORP family proteins including OSBP may play an important role in maintaining sterol balance in cells of the central nervous system.Human OSBP was found to function as a cholesterol-binding scaffolding protein coordinating the activity of two phosphatases(serine/threonine phosphatase,PP2A,tyrosine phosphatase,HePTP)to control the extracellular signal-regulated kinase(ERK)signaling pathway.In addition,it has been reported that phosphorylated ERK can promote neurite outgrowth.However,the role of OSBP in the neurite outgrowth is still unknown.Based on the current status,we are not only committed to exploring the target gene of miR-124a in promoting neurite outgrowth,but also try to find the mechanisms.First of all,we used the online bioinformatical tool(TargetScan)to predict the target genes of miR-124a and screen the candidate genes by reviewing the relevant studies.We chosen OSBP as the candidate gene that has three conserved target sites of miR-124a in OSBP 3'UTR.Next,in order to verify that miR-124a binds to the candidate gene OSBP through seed sequence,we got the full-length sequences of OSBP 3'UTR(mouse origin)from Genbank website and used the software Primer Premier 6.0 to design PCR primer sequences.Then the genomic DNA of N2a cells was used as the template for PCR to construct the miR-124a overexpression plasmid hU6-miR-124a-CMV-EGFP(U6-124),and U6-124 plasmid was used as the template to construct miR-124a mutant plasmid hU6-miR-124amut-CMV-EGFP(U6-124mut),AAGG was mutated as TTCC.We constructed OSBP luciferase reporter plasmid that containing all target sites of miR-124a in OSBP 3'UTR,and OSBP 3'UTR plasmid was used as the template to construct OSBP 3'UTR mutant plasmid(OSBP 3'UTRmut),GCCT was mutated as CGGA.Then we co-transfected HEK293 cells with these plasmids by using LipofectamineTM2000.This experiment included two groups,one group being OSBP 3'UTR with U6-124,U6-124mut or control plasmid U6,respectively;another group being OSBP 3'UTRmut with U6-124,U6-124mut or control plasmid U6,respectively.We used Dual-Luciferase(?)Reporter Assay System Kit to detect the Renilla luciferase signal after 24 hours transfection.The Renilla luciferase signal was normalized to the firefly luciferase signal.The luciferase reporter assay showed that the luciferase activity of OSBP 3'UTR co-transfected with U6-124mut[(93.97± 10.19)%]was significantly higher than OSBP 3'UTR co-transfected with U6-124[(50.63±9.58)%,n=4,P=0.009].And,the luciferase activity of OSBP 3'UTRmut co-transfected with U6-124[(80.19±10.56)%]was significantly higher than OSBP 3'UTR co-transfected with U6-124[(50.63±9.58)%,n=4,P=0.005].There was no significant difference between OSBP 3'UTR co-transfected with U6[(100.00±8.78)%]and OSBP 3'UTR co-transfected with U6-124mut[(93.97±10.19)%,n=4,P=0.936].These results indicated that miR-124a could bind specifically to the 3'UTR sequences of OSBP to inhibit gene expression,implying that OSBP maybe is the direct target gene of miR-124a.To further test whether miR-124a can repress the expression of OSBP,the miR-124a overexpression plasmid or the control plasmid U6 was transfected to 293 cells by using LipofectamineTM2000.96 hours after transfection,the protein expression of OSBP was detected by western blot analysis.We found that the OSBP protein level significantly reduced in cells tranfected with U6-124,when compared with the cells transfected with control plasmid U6,further demonstrating that miR-124a represses the expression of OSBP.Next,we used a final concentration of 20 ?mol/L retinoic acid-induced N2a cell differentiation model to verify the role of miR-124a in the neurite outgrowth.The cell morphology was observed by fluorescence microscopy 24 hours after retinoic acid induction.The results showed that the neurite length of the cells transfected with miR-124a overexpression plasmid U6-124(n=24,146.82±19.06)was significantly higher than that of the cells transfected with the control plasmid U6(n=20,98.31±19.53,P<0.001).The percentage of the cells with neurite length more than twice the cell body in the U6-124 group[n=24,(45.23±7.65)%]was also significantly higher than the control[n=20,(11.11 ±5.05)%,P<0.001],suggesting that miR-124a can significantly increase the neurite outgrowth of differentiated N2a cells.We used the shRNA-mediated gene silencing approach to explore the role of OSBP in the neurite outgrowth.The interference sequence of OSBP was chosen by referring to the efficient sequence in the previous studies.The shRNA-mediated plasmid pGPU6-shOSBP-CMV-GFP(shOSBP)was constructed by Gemma Company in Shanghai.N2a cells were transfected with shOSBP plasmid or shcontrol plasmid by using LipofectamineTM2000,respectively.Then we used a final concentration of 20 ?mol/L retinoic acid to induce differentiation.The cell morphology was observed by fluorescence microscopy 48 hours after retinoic acid induction.The results showed that the neurite length of cells transfected with shRNA-mediated plasmid shOSBP(n=34,189.03±68.47)was significantly higher than that of the cells transfected with the shcontrol plasmid(n=40,134.81 ±36.27,P<0.001).The percentage of the cells with neurite length more than twice the cell body in shOSBP group[n=35,(40.26±10.79)%]was also significantly higher than the control[n=41,(16.13±6.32)%,P<0.001].These results indicated that OSBP could significantly inhibit the neurite outgrowth of differentiated N2a cells.To further investigate the role of OSBP in the neurite outgrowth,we got the full-length sequences of OSBP mRNA(mouse origin)from Genbank website and used the software Primer Premier 6.0 to design cloning PCR primer sequences.Then we used the cDNA(100ng/?l)of N2a cells as the template for PCR to construct the OSBP overexpression plasmid.MiR-124a overexpression plasmid U6-124 was co-transfected with OSBP overexpression plasmid OSBP-Cherry or control plasmid pmCherry-N1 by using LipofectamineTM2000.The cell morphology was observed by fluorescence microscopy 24 hours after retinoic acid induction.The results showed that the neurite length of cells co-transfected with U6-124 plasmid and pmCherry-N1 plasmid(n=7,171.15±30.32)was significantly higher than that of the cells co-transfected with U6-124 plasmid and OSBP-Cherry plasmid(n=7,132.09±28.97,P=0.015).So our results displayed that OSBP overexpression inhibited miR-124a in promoting neurite outgrowth,and further demonstrated that OSBP inhibited neurite outgrowth.To investigate the role of OSBP in the neurite outgrowth of neurons,the primary cortical neurons were prepared from embryonic days 18-19 C57 mouse pups.Then the neurons were electrotransfected with OSBP overexpression plasmid OSBP-Cherry or control plasmid pmCherry-Nl.The cell morphology was observed by fluorescence microscopy after transfection.The results showed that 48 hours after transfection,the neurite length of neurons transfected with pmCherry-Nl plasmid(n=19,112.41 ±44.87)was significantly higher than that of the neurons transfected with OSBP-Cherry plasmid(n=15,69.48±23.19,P=0.002),similar results were also seen at 72 hours and 96 hours after transfection.So our results revealed that OSBP overexpression inhibited the neurite outgrowth in primary cultured neurons.Previous studies have reported that miR-124a expression levels have significant changes during mouse brain development,with the high level at embryonic day 14 and day 17,reaching a plateau in postnatal.Therefore,in order to investigate whether the expression of OSBP in brain changes,we first checked the mRNA expression of OSBP during different developmental stages of C57 mouse cortex.C57 mouse cortices were collected from embryonic day 14,day 18,and postnatal day 1,7,30,60.There are 3 to 4 mouse cortices in every group.We extracted RNA of cortical tissues and used qRT-PCR to detect the mRNA levels of OSBP gene.The results showed that the expression levels of OSBP mRNA tended to decrease during cortical development.The expression at postnatal day 1(n=3,0.66±0.34)was less than the embryonic day 14(n=3,1.021±0.22,P=0.05),but the difference was not significant.However,the expression at postnatal 7 day(n=4,0.52±0.20)was significantly lower than the embryonic day 14(n=3,1.02±0.22,P=0.006),and the expression remained at low level steadily into adulthood.Our data showed that OSBP expression decreased during brain development,which is consistent with the increase of miR-124a.However,whether OSBP is regulated by miR-124a in neurons still needs further studied.In conclusion,our study found that miR-124a promoted neurite outgrowth during N2a cell differentiation.MiR-124a combined with 3'UTR sequences of OSBP gene to inhibit it expression,so OSBP might be the direct target gene of miR-124a.In addition,blocking OSBP by shRNA enhanced neurite outgrowth,while overexpressing OSBP suppressed miR-124a-mediated neurite outgrowth.Moreover,we found that OSBP expression decreased during brain development.Our data indicated that in N2a cell differentiation model,miR-124a might promote neurite outgrowth by inhibiting OSBP expression.In mammal brain development,whether miR-124a promotes neurite outgrowth by inhibiting OSBP needs to be further studied.