Function And Mechanism Of MiR-31 In Spinal Cord-derived Neural Stem Cells And Their Differentiation Into Motor Neuron-like Cells

Objective: After spinal cord injury(SCI),damaged neurons rarely repaired themselves at the injury site,leading to severe dysfunction.The discovery of neural stem cells(NSCs)provided a promising feasibility option for the radical treatment of spinal cord injury,but studies had found that nearly all transplanted neural stem cells differentiated into glial cells in mouse spinal cord injury models.The reason for this was mainly due to the insufficient clarity of the mechanistic understanding of the differentiation of neural stem cells into motor neurons(MNs)to actively perform directed induced differentiation.The reason was mainly due to the insufficient understanding of the differentiation mechanism of neural stem cells into MNs,which made it impossible to actively induce differentiation.Studies had shown that miRNA could precisely regulate the expression of target gene m RNA by forming RNA induced silencing complex,acting on the 3'UTR region of target gene m RNA,silencing the previous active molecules during cell differentiation,and establishing clear spatiotemporal expression boundaries.The changes of one key miRNA expression might ultimately lead to global alterations in the gene expression profile for a cell,thereby providing reassurance for stable conversion of cell fate.It had been confirmed that miRNA was involved in a series of activities such as self-renewal,proliferation and differentiation of neural stem cells.To search for some miRNAs that played a key role in the differentiation of NSCs into MNs,our group previously investigated the differential expression of miRNAs between spinal cord derived NSCs and MNs and found that miR-31 expression in NSCs was more than 90 fold higher than that in MNs.In addition,there were evidence that miR-31 could promote the amplification of mammary stem cells and intestinal stem cells.All these could be inferred that miR-31 was a key stemness maintenance gene in NSCs and might play a negative regulatory role in the differentiation of NSCs into MNs,so the study of the specific role of miR-31 in NSCs could help us further understand the mechanism of NSCs and their differentiation into MNs.Methods: In this study,we explored the role of miR-31 in NSCs and their differentiation into MNs through molecular biology experiments and bioinformatics,and preliminarily studied the effect of miR-31 si RNA drugs on neural stem cells in vitro.1.Observe the expression changes of NSCs and MNs-related genes,through interfering or overexpressing miR-31 in NSCs.And conduct bioinformatics analysis of predicted target genes and validate target genes of miR-31 to understand the function of miR-31 in NSCs.2.Investigate the mechanism of miR-31 by studying the differential gene expression at the early stage of spinal cord derived NSCs differentiation after miR-31 interfering by transcriptome sequencing technology.3.Investigate the stemness maintenance mechanism of miR-31 by analyzing the differential m RNA and miRNA expression profiles in the early stage of spinal cord derived NSCs after overexpressing miR-31 by transcriptome sequencing technology.And validate the miR-31 target gene by using dual luciferase reporter assay.4 Investigate the effects of miR-31 si RNA drugs on proliferation and differentiation of neural stem cell in vitro.Results: 1.The expression of MNs-related genes ChAT,Hb9,Nkx6.1,Nkx6.2,Isl1,Lhx3 and Olig2 were increased to various degrees after miR-31 interfering,while the expression of Nestin,a neural stem cell marker,was decreased.These indicated that interfering with miR-31 expression could induce NSCs differentiation into MNs-like cells.However,the MNs-related genes expression was down regulated when miR-31 was overexpressed,while Nestin expression was increased,which suggested that overexpression of miR-31 could maintain NSCs stemness and inhibit their differentiation.2.After miR-31 interfering,there were 10 up-regulated genes and 37 down-regulated genes(compared with the interference control group),and 746 up-regulated genes and 549 down-regulated genes(compared with the NSCs group)in the miR-31 interfering group.The function of up-regulated genes was mainly related to the differentiation of NSCs,especially into motor neurons.However,the down-regulated genes were closely related to the selfrenewal and proliferation of NSCs.3.After miR-31 overexpressing,there were 13 up-regulated genes,22 down-regulated genes,169 up-regulated miRNAs and 169-down regulated miRNAs(compared with the overexpression control group),and 719 up-regulated genes and 579 down-regulated genes(compared with the NSCs group)in the miR-31 overexpressing group.The function of down-regulated genes and miRNA was mainly related to the differentiation of NSCs.However,the down-regulated genes and miRNA were closely related to the NSCs stemness maintenance.And confirmed that Rerg was a target gene of miR-31 by dual luciferase reporter assay.4.The miR-31 si RNA drugs could be directly transfected into cells without transfection reagent.Among them,agomir drugs could promote the proliferation of neural stem cells,and antagomir drugs could promote the differentiation of neural stem cells into MNs-like cells.Conclusion: miR-31 was a key stemness maintenance gene in NSCs,mainly by affecting the expression of numerous miRNAs and m RNAs associated with proliferation and differentiation,and played a negative regulatory role in the differentiation of NSCs into motor neurons.miR-31 si RNA drugs could enter cells without transfection reagent,and exert the same effect as common si RNA inhibitors.This study provided an effective candidate target for effectively inducing neural stem cells to differentiate into motor neurons,and also laid a preliminary foundation for the effective application of miR-31 si RNA drugs in the clinical treatment of spinal cord injury and motor neuron diseases.