| Mesenchymal stem cells(MSCs)are mesoderm derived cells that can differentiate into many types of tissue cells.It mainly existed in the connective tissue and interstitial tissue,and the bone marrow tissue has the most.Several studies have reported MSCs can migrate to the damaged area to repair injured tissue.Cytokines,such as HGF,which is secreted from tissues around injury and inflammation sites could promote the directed motility of MSCs to the injured area.However,only a small number of transplanted MSCs successfully reached the injured tissues after injection,which severely limited the clinical applications of MSCs.Consequently,it’s essential to improve the migratory ability of MSCs,increase the number of transplanted cells to the injured or diseased sites.Micro RNAs(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 m RNAs for cleavage or translational repression.With the further research of miRNAs,miRNAs are involved in a broad range of cellular processes,including cellular proliferation,differentiation and migration.However,relatively few studies have examined miRNA function in MSCs migration.Previous results in our lab demonstrated that HGF induced migration of MSCs,the MSCs in varying neural differentiation states affected the ability of directional migration.Besides,miR-26 b promotes the migration of MSCs toward HGF.So we first determined the differential expression of miR-26 b of MSCs under varying neural differentiation states.We found that miR-26 b expression was different.Then,we investigated the chemotactic responses of MSCs in different neural differentiation states toward HGF.Results showed that HGF for 5 h induced miR-26 b expression rising in undifferentiated MSCs and 24-h preinduction state,but not in 5-h induction and 18-h maintenance statess.Taken together,we speculate that miR-26 b may be involved in the migration of MSCs toward HGF in varying neural differentiation states.We transiently infected miR-26 b adenovirus into MSCs of different neural differentiation states to create a gain-of-function behavior in cells.The effect of miR-26 b on migratory capacity of MSCs was assessed by the Boyden chamber assay.The results demonstrated that overexpression of miR-26 b caused robust chemotactic migration toward HGF of undifferentiated,24-h preinduction and 18-h maintenance MSCs.Inhibition of miR-26 b function reduced chemotactic migration toward HGF of all differentiating MSCs.Dunn chamber assay found that miR-26 b significantly increased the migratory velocity of undifferentiated and 24-h preinduction MSCs,but not 5-h induction and 18-h maintenance states,miR-26 b does not altered the FMI of all differentiating MSCs toward HGF.In summary,we identified that miR-26 b modulates migration of MSCs in varying differentiation states.PIP3 dephosphorylated by the lipid phosphatase activity of PTEN,which blocked phosphorylation of Akt,suppressed PI3K/Akt signaling pathway.So,whether PI3K/Akt signaling pathway modulates miR-26 b promoting the migration of MSCs in varying differentiation states toward HGF.In order to verify the relationship between PTEN,miR-26 b and PI3K/Akt,we constructed PTEN recombinant adenovirus vector.The recovery experiment further confirmed that miR-26 b targeted PTEN,which promoted the migration of MSCs toward HGF.In addition,we found that overexpression of miR-26 b significantly promoted the phosphorylation of Akt in varying differentiation states,while the downexpression of miR-26 b significantly inhibited the phosphorylation level of Akt.And altering the expression of miR-26 b did not affect the expression of differentiating MSCs SAPK/JNK,ERK1/2and p38 MAPK phosphorylation.The migration of MSCs in varying differentiation states toward HGF was significantly inhibited after treated MSCs with LY294002.These results indicated that miR-26 b affect the migration of MSCs toward HGF in varying differentiation states through PI3K/Akt signaling pathway,not MAPKs signaling pathway.Mi R-26 b promoted the migration of MSCs toward HGF in varying differentiation states by activating Akt,but the inhibition of PI3K/Akt could not completely inhibit the promoting effect of miR-26 b on cell migration.These may indicated that other signaling pathways involved in the regulation of MSCs migration by miR-26 b,such as,FAK 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-26 b on the formation of FA and reorganization of F-actin.Immunocytochemical analysis showed that upregulating of miR-26 b can promote cells formed a single large lamellipodia at one end and increase the number of subtle new focal adhesion.Overexpression of miR-26 b partial returned migration toward HGF,these may indicated that other target genes involved in the regulation of MSCs migration by miR-26 b.Thus,putative miR-26 b targets were predicted using target prediction programs,Target Scan and miRDB.We found that GSK-3β and ROCK1 3 ’UTR region has miR-26 b binding sites.Moreover,RT-q PCR analysis showed that GSK-3β and ROCK1 m RNA levels were significantly downregulated in miR-26b-transfected cells,which descripted miR-26 b may target GSK-3β and ROCK1.After Y27632 treatment with MSCs 30 min,we observed changes in cell morphology by TRITC-Phalloidinmin immunofluorescence staining.The results showed that the polarity of the cells was significantly enhanced,cell area changes were not significant,the easier the formation of lamellipodia at the end of cell.However,its specific mechanism remains to be confirmed.In summary,we demonstrated that miR-26 b promoted the directional migration of MSCs in varying differentiation states by regulating the PI3K/Akt signaling pathway.In addition,miR-26 b promoted cells formed a single large lamellipodia at one end,increased the number of subtle new FA and enhanced FA to the cell periphery distribution.All these results invite a speculation that a manipulation of the level of miR-26 b in transplanted MSCs may benefit the therapeutic efficiency of transplantation. |
