The Experimental Study Of The Roles Of MicroRNAs On Human Hair Follicle-derived Neural Crest Stem Cells Differentiation Into Schwann Cells

Nowadays, implant-supported denture has been the first choice of the patientswith dentition defect or edentulous jaw, with the rapid development of materialsscience and oral implants. Because of the lack of periodontal ligament, implantdenture has less sensitivity to feel the load than natural teeth, so we expect implantdenture can be more sensitive to the occlusal force.This goal can be achieved by thestudies of nerve regeneration.Studies have shown that Schwann cells (SCs) can promote the restoration andregeneration of peripheral nerve tissue. SCs are glial cells in the peripheral nervoussystem. After peripheral nerve injury, SCs engulfed axons and myelin debris, andform the band of Bungner with rapidly proliferating. At the same time, they secreteneurotrophic factors, extracellular matrix and adhesion molecules to guide axonalgrowth, to maintain the viability of neurons and to promote the reinnervation process.Neural crest stem cells (NCSCs) originated from the structure of embryonic neuralcrest, with the capacity of self-renewal and multilineage differentiation. Hair folliclederived NCSCs are Schwann cell precursors which are the ideal source of SCs.MicroRNAs(miRNAs) are a class of non-coding single-stranded small RNAmolecules of18–25nucleotides that can bind to the3’UTR of the mRNA moleculesand regulate the protein expression of target gene. Approximately1,600humanmiRNAs have been identified to participate in various physiological andpathophysiological processes, including stem cell differentiation, tumor formation andmetastasis, cell apoptosis, inflammation and embryonic development.In this study, we isolated neural crest stem cells from human hair follicles, andinduced them differentiated into Schwann cells. We performed a global miRNAanalysis with the application of microarray assay to reveal and identify characteristicpatterns in the differentiation of NCSCs into Schwann cells. We also explored the function and mechanism of identified miRNAs in the process of differention. Ourresults provide theoretical basis for clinical application of miRNAs and NCSCs in thefuture.1.Induction of human hair folliclle-derived neural crest stem cells differentiatedinto Schwann cells.Objective: To isolate and culture human hair follicle-derived neural crest stemcells, and induce them to express Schwann cell markers.Methods: Hair follicles were isolated from the human deciduous epidermis, thenwere treated with0.25%trypsin/EDTA. The resultant cells were cultured in hESCmedium with4ng/mL basic fibroblast growth factor. NCSCs were screened usingflow cytometric cell sorting. HNK1and p75double positive cells were collected.NCSCs were induced to differentiate into SCs, using MesenPRO medium containing20ng/mL neuregulin-1for a total of28days. Flow cytometry was used to investigatethe cell differentiation. S100and GFAP were detected, which were Schwann cellmarkers.Results: A small number of shuttle-shaped cells appear around the hair folliclesat48hours after culture in medium. The HNK1and p75double positive cells aresorted out by flow cytometric cell sorting and accounted for10%of all cells. AfterNCSCs were cultured in culture medium for about5days, small typical clumps ofstem cells formed. Flow cytometric cell sorting showed that at day30of induction,63.8±3.7%of cells expressed S100and72.6±4.9%of cells expressed glial fibrillaryacidic protein (GFAP). These results indicate that after induction by neuregulin-1,NCSCs differentiated into SC-like cells.Conclusion: We successfully isolated and purified human hair follicle-derivedNCSCs and they formed small typical clumps of stem cells. NCSCs could be induceddifferentiated into Schwann cells with neuregulin-1stimulation, expressing specificmarkers S100and GFAP.2. MicroRNA screening and verification of the differentiation from NCSCs intoSchwann cellsObjective: To screen miRNAs which participate in the differentiation fromNCSCs into Schwann cells. To verify the specific miRNA expression in thedifferentiation, and to observe the influence of miRNA in the differentiation.Methods: Screening miRNAs paticipated in the differentiation through microarray assay, S16cells/NCSCs as a group and induced40days NCSCs/undifferentiated NCSCs as another group. Selecting miR-21as a target,0and10,20,30,40days after induced differentiation，NCSCs were collected and intracellularmiR-21expression was detected using qRT-PCR. The agonist agomir-21andantagonist antagomir-21were added to NCSCs medium, which artificially regulatedthe expression level of miR-21, to detect S100and GFAP expression by flowcytometry and qRT-PCR.Results: Microarray assay shows, compared with undifferentiated NCSCs, therewere76kinds of miRNAs up-regulated more than4times in NCSCs which induced40days,and89kinds down-regulated more than4times. Quantitative RT-PCR testsshowed that miR-21expression was detected at different time periods of induction,and the expression was about3times higher at day10of induction compared to thelevel prior to induction and increased gradually thereafter (P <0.05). This resultdemonstrates that miR-21plays a regulatory role during NCSC differentiation intoSchwann cellsAfter transfected with miR-21agonist (agomir-21) or miR-21antagonist(antagomir-21), results showed that after neuregulin-1induction,75.3±5.6%ofagomir-21-transfected NCSCs expressed both S100and GFAP simultaneously, whichwas significantly higher than that in the corresponding negative control (agomir-NC)group (64.2±4.1%, P <0.05); after neuregulin-1induction,41.1±3.2%of antagomir-21-transfected NCSCs expressed both S100and GFAP simultaneously, which wassignificantly lower than that in the corresponding negative control (antagomir-NC)group (62.0±2.7%, P <0.05).In the quantitative RT-PCR assay,results showed that after neuregulin-1induction, S100mRNA expression in the agomir-21group and antagomir-21groupwere significantly higher and lower than that in the agomir-NC group (P <0.05) andantagomir-NC group (P <0.05), respectively; after neuregulin-1induction, GFAPmRNA expression in the agomir-21group and antagomir-21group were significantlyhigher and lower than that in the agomir-NC group (P <0.05) and antagomir-NCgroup (P <0.05), respectively. These findings demonstrate that miR-21can promotethe differentiation of NCSCs into SCs.Conclusion: The microarray assay showed miR-21expression was up-regulatedin induced NCSCs.The qRT-PCR result verified the upregulation of miR-21.The agonist and antagonist of miR-21can regulate the differentiation of NCSCs,and miR-21can promote this process.3.MiR-21promotes hair follicle-derived NCSCs differentiation into Schwanncells via targeting SOX2Objective: To investigate the mechanisms of miR-21regulation, to find thetarget of miR-21and to clarify the relationship between miR-21and SOX2.Methods: We retrieved the information of miR-21target in Pictar,Targetscanand miRBase databases. The SOX2mRNA3’UTR was inserted in the luciferasereporter system (SOX2-3’ UTR-wt) and simultaneously the mutational SOX2mRNA3’ UTR was also inserted into this reporter system (SOX2-3’UTR-mut). miR-21wastransfected into HEK-293cells together with luciferase empty vector, SOX2-3’UTR-wt or SOX2-3’UTR-mut to investigate whether miR-21can bind with SOX2mRNA3’UTR.Transfected NCSCs with agomir-21and antagomir-21to detect SOX2proteinand mRNA expression by western blot and qRT-PCR at48h. To investigate whetherSOX2participates in miR-21promotion of stem cell differentiation, SOX2wereknocked out from NCSCs. NCSCs and SOX2-KD-NCSCs were divided intoantagomir-21and antagomir-NC groups. After induction for40days, thedifferentiation of NCSCs was detected by flow cytometry and qRT-PCR.Results: We found that a7-mer-long sequence at the3’-UTR of SOX2mRNAwas matched to miR-21in the internet database. Results showed that the fluorescenceintensity in the SOX2-3’UTR-wt group was significantly lower than that in thenegative control (miR-con) group. However, there was no significant difference ininflorescence intensity between SOX2-3’UTR-mut group or empty vector group andmiR-con group. These findings suggest that miR-21can bind to the3’UTR of SOX2mRNA.The western blot results showed that agomiR-21can significantly decreaseintracellular SOX2expression (P <0.05), while antagomiR-21can significantlyincrease intracellular SOX2expression (P <0.05). SOX2mRNA expression was notinfluenced by agomiR-21or antagomiR-21. These findings suggest that miR-21canregulate SOX2protein expression at the post-transcriptional level by binding to the3’-UTR of SOX2mRNA.We silenced SOX2expression in NCSCs using the lentiviral shRNA approachand compared these SOX2-knocked out NCSCs(SOX2-KD-NCSCs) with those non- interfered NCSCs (control) to investigate the role of SOX2in miR-21regulation ofNCSC differentiation. Flow cytometry and quantitative RT-PCR results showed thatantagomiR-21-transfected NCSCs exhibited a significantly decreased differentiationcapacity than antagomiR-NC-transfected cells (P<0.05). However, there was nosignificant difference in the differentiation capacity between antagomiR-21-transfected SOX2-KD-NCSCs than antagomiR-NC-transfected SOX2-KD-NCSCs.These results demonstrate that SOX2knockout can block the effect of antagomir-21against SC differentiation, indicating that SOX2is one of downstream pathways bywhich miR-21regulates the differentiation of NCSCs.Conclusion: MiR-21can bind to the3’UTR of SOX2mRNA to regulate SOX2protein expression at the post-transcriptional level. SOX2knockout can block theeffect of antagomir-21against SC differentiation, indicating that SOX2is one ofdownstream pathways by which miR-21regulates the differentiation of NCSCs.