MiR-124 Modulates The Migration Of Mesenchymal Stem Cells Toward HGF By Regulating Wnt/β-catenin Signaling Pathway

Mesenchymal stem cells (MSCs) are multipotent cells with the capacity to differentiate into many types of tissue cells, including osteoblasts, chondrocytes, adipocytes and neural cells. Cultured MSCs are low immunogenicity and capable of homing thus have emerged as a promising source of cells for repair of a number of damaged tissues. Several studies have reported the abilities of growth factors and cytokines such as hepatocyte growth factor (HGF) which is secreted from tissues around injury and inflammation sites could promote the directed MSC motility to the injured area. However, only a small population of transplanted MSCs successfully reached the injured tissues after injection, which severely limit the clinical applications of MSCs. Therefore, to study the molecular events associated with the migration of MSCs will help to improve the application of MSCs as therapeutic vehicles.MicroRNAs (miRNAs) are small non-coding RNAs of 20-24 nucleotides that silence gene expression post-transcriptionally by binding to the 3’untranslated regions (3’UTRs) of target mRNAs for cleavage or translational repression. Hundreds of miRNAs have been identified, most of which are predicted to target abundant of mRNAs, suggesting that miRNAs may function as part of an extensive gene regulatory network. Increasing numbers of studies have demonstrated that miRNAs are involved in a broad range of cellular processes, including the regulation of embryonic development, cellular proliferation, differentiation and migration. However relatively few studies have examined miRNA function in MSCs. The aim of this study is to investigate whether miR-124 modulates the chemotactic migration of MSCs and modulates Wnt/β-catenin signaling pathway.Previous results in our lab demonstrated that HGF induced migration of MSCs in a dose-dependent manner, with the maximum migration at 50 ng/ml. So we first determined the differential expression of miR-124 after 50 ng/ml HGF stimulation. We found that miR-124 expression was downregulated after HGF stimulation, which may be involved in modulating the migration of MSCs toward HGF. We transiently transfected miR-124 mimic into MSCs, to create a gain-of-function behavior in cells. The effect of miR-124 on migratory capacity of MSCs was assessed by the wound-healing and Boyden chamber assay. All the results demonstrate that up-regulating of miR-124 can reduce the migratory capacity of MSCs, while down-regulating miR-124 by miRNA inibitor can significantly improve MSC migration activity. To further unravel the molecular mechanisms involve in miR-124-mediated effects in MSCs, identifying the putative targets of miRNAs is necessary. Thus, putative miR-124 targets were predicted using target prediction programs, TargetScan. Our analysis revealed that FZD4 and LRP6 were two potential targets of miR-124. We conducted a 3’UTR lucifease assay and observed that the luciferase activity were decreased after co-transfection of miR-124 with 3’UTR vectors containing FZD4 and LRP6 target sequence. Moreover, qRT-PCR and Western blot analysis showed that FZD4 mRNA and protein levels were significantly downregulated in miR-124-transfected cells. However, LRP6 mRNA expression was slightly decreased and had no statistical difference in over-expressed miR-124 group, LRP6 protein levels were significantly reduced, indicated the repression of LRP6 expression occurred post-transcriptionally.One of the major signal transduction pathways that has been associated with various stem cell attributes is the Wnt/β-catenin signaling. The Wnt/β-catenin signaling pathway is activated by binding of Wnt ligands to a family of seven-pass transmembrane Frizzled (FZD) receptors and single-pass transmembrane LRP5/6 co-receptors, leading to stabilization of cytosolic β-catenin, which accumulates and travels to the nucleus to form complexes with TCF/lymphoid enhancer factor (LEF) transcription factor leading to the transcription of target genes. Because miR-124 negatively regulates FZD4 and LRP6, we hypothesized that miR-124 negatively affects Wnt/β-catenin signaling. To test the hypothesis, MSCs were transiently transfected with the Wnt signaling reporter TOPFlash or the control FOPFlash, along with miR-124 mimic or NC. We found that over-expression of miR-124 inhibited the TOP flash but not the FOP flash reporter in MSCs after Wnt3a stimulation. Immunocytochemical analysis showed that up-regulating of miR-124 can disturbed nuclear translocation of (3-catenin in MSCs, while down-regulating miR-124 by miRNA inibitors can significantly induced translocation of the cytosolic β-catenin into the nucleus. Moreover, by the ectopic expression of miR-124, we found that the levels of activated β-catenin (ABC) were decreased and the levels of phosphorylated β-catenin at its Ser33/37/Thr41 were increased in MSCs. Using real-time PCR, we found that the expression of RUNX2 and c-Myc were decreased in MSCs over-expressing miR-124. These results suggest that miR-124 inhibits the Wnt/β-catenin signaling in MSCs.To further study whether the inhibitory effects of miR-124 on MSC migration is associated with the regulation of Wnt/β-catenin signaling pathway, we used Wnt3a and FH535, which is a synthetic inhibitor of the Wnt/p-catenin signaling by suppressed TCF4 transcription. The results showed that over-expression of miR-124 markedly reduced the Wnt3a-induced migration of MSCs. Moreover, blocking Wnt signaling by FH535 almost fully abolished miR-124 inhibition induced the migration of MSCs towards HGF. These results indicated that the MSC migration suppressive function of miR-124 might be through the modulation of Wnt/β-catenin signaling pathway.Cell migration consists of several temporally and spatially coordinated events, including protrusion of the leading edge to form lamellipodium or filopodium, adhesion of the leading edge to the substrate, translocation of cell body, and release of the trailing edge. So we finally detected the effects of miR-124 on the formation of FAs and reorganization of F-actin. Immunocytochemical analysis showed that up-regulating of miR-124 can decrease the number of FAs and disturbed the formation of lamellipodia.In conclusion, we demonstrated that ectopic expression of miR-124 reduced the HGF-induced chemotactic migration of MSCs. More importantly, we found that miR-124 directly target FZD4 and LRP6 genes, which result in inhibition of Wnt/β-catenin signaling. All these results invite a speculation that a manipulation of the level of miR-124 in transplanted MSCs may benefit the therapeutic efficiency of transplantation.