The MiRNA Changes In Differentiation Of BMSCs Induced By Schwann Cell Into Neuron

【Objective】Spinal Cord Injury(SCI)is a serious traumatic disease,and the incidence rate increases year by year with the rapid development of China’s economy.Although many countries have conducted extensive research in the field of spinal cord injury,there has been no breakthrough in effective treatment for spinal cord injury.At present,the most promising treatment for spinal cord injury is cell transplantation,in the application of Schwann Cells(SCs)and Bone Mesenchymal Stem Cells(BMSCs).Bone marrow mesenchymal stem cells(BMSCs)have multi-directional differentiation ability,and more and more studies focused on the differentiation of BMSCs into neurons.Under the induction of Schwann cells(SCs),BMSCs can differentiate into neural-like cells.In this study,we will screen and identify different expressed microRNAs in bone marrow mesenchymal stem cells(BMSCs)induced by Schwann cells(SCs)conditional medium,and to explore their targets and related pathways involved in the differentiation of BMSCs into neuron-like cells.【Method】The primary BMSCs were isolated from femoral and tibia bones from adult Wistar rats,then expanded until passage 3 for identification using flow cytometry and differentiation experiment.and the primary SCs were isolated from the bilateral Sciatic nerves,identifying the purity of the obtained SCs was by S-100 immunofluorescence staining after the third passage.The experiment contained two groups: Schwann cell conditioned medium(SCs-CM)group and normal medium,For neuronal differentiation of BMSCs using Schwann cell conditioned medium(SCs-CM)induction,the co-culture of BMSCs and SCs was applied in this experiment.Neuronal differentiation of BMSCs was induced by Schwann cell conditioned medium(SCs-CM),which observed at the different time point under the microscope.The quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to analysis the relative mRNA expression associated with neural markers in BMSCs.MicroRNA analyses was used to identify changed microRNA.Target genes were predicted with Targetscan,microRNAorg and PITA,followed by Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.【Result】The BMSCs was isolated from femoral and tibia bones in adult Wistar rats,and the undifferentiated cells at P3,P4,and P5 were used for flow cytometry experiment to test the expression of surface marker(CD34,CD45,and CD29,and CD90).BMSCs showed positive expression for a CD29 and CD90.On the contrary,BMSCs were clearly almost negative for the haematopoietic markers like CD34,CD45.The bilateral Sciatic nerves of the rats were used to isolate the SCs,and the P3 cells were used to examine for the identification through S-100/DAPI immunofluorescence staining.the morphology of cells almost was spindle type,and 95.7±0.8% cells were positive for S100.Schwann cell conditioned medium(SCs-CM)was used to induce BMSCs differentiated into neuron-like cells,and the effect of SCs-CM on the differentiation of BMSCs was observed at the different time point under the microscope.The shape of BMSCs cells was changed into neuron-like cells.The cell body of BMMSCs gradually shrinks and becomes bright,and there are several neurites were formed,and eventually BMSCs formed a neuron-like cells.The quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to examine gene expression levels of Nestin and MAP2 in BMMSCs.In SCs-CM group,the Nestin were up-regulated from 1-3day and down-regulated from 3-7days,and the MAP2 was increased gradually from 1-7days compared with control group.After 7days induction,MicroRNA analyses was used to identify changed microRNA.There were 83 significantly different expressed miRNAs between two groups.and 77 miRNAs were upregulated,and 6 miRNAs were down regulated.These DE-miRNAs were used to figure out the target genes,and further bioinformatics analysis.Gene Ontology(GO)analysis showed that the miRNAs target genes enriched in neuron projection development,regulation of axonogenesis and positive regulation of cell proliferation.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis pathway showed Hippo,Wnt,TGF-β and Hedgehog signaling pathway were the potential acting pathways associated with neural differentiation in BMSCs.【Conclusion】In this study,SCs could induce BMSCs differentiated into neuron-like cells.the expressing profile of miRNA in BMSCs induced differentiate into neuron-like cells were investigated,and 83 DE-miRNAs were obtained.The results of GO and KEGG analysis showed that the significantly changed miRNAs targets enriched in several neuronal differentiation relative pathways,including Hippo signaling pathway,Wnt signaling pathway,TGF-β signaling pathway and Hedgehog signaling pathway.This study provided the miRNAs profiling,and further indicated the key miRNAs and pathways in BMSCs neural differentiation.These results will provide a theoretical basis for the treatment of neurological diseases by combined cell transplantation.