MiR-26b Regulates The Migration Of Mesenchymal Stem Cells By Targeting ROCK1

Mesenchymal stem cells(MSCs)are multipotent mesoderm-derived stromal cells with the ability to self-renew and differentiate into a variety of tissue cells.Due to their low immunogenicity,simple isolation,and capability of homing to injured or diseased sites,MSCs can be used to treat various tissue damages,but the very limited number of cells that migrate to the damaged region strongly restricts their therapeutic applications.Therefore,better understanding of the mechanisms that regulate the migration of MSCs and discovering conditions that elevate their migration ability,will help to increase their homing ability and improve their therapeutic efficacy.In this study we further investigated the mechanism underlying miR-26b's promotion of MSC migration through targeting Rho-associated kinase 1(ROCK1).Firstly,we found that upregulating the expression of miR-26 b by transfection of miR-26 b mimic significantly reduced ROCK1 at the m RNA levels,however,inhibiting the expression of miR-26 b by transfection of miR-26 b Inhibitor(miR-26 b I)significantly increased ROCK1 at the m RNA levels.Western blot showed that upregulation of miR-26 b significantly decreased the expression of ROCK1 at the protein level,however,inhibition of miR-26 b significantly increased the expression of ROCK1 at the protein level.These results indicate that miR-26 b negatively regulates the expression of ROCK1.Further,We studied whether ROCK1 is a direct target of miR-26 b.We obtained the wild-type 3'UTR fragment of ROCK1 containing the conserved binding site of miR-26 b by PCR and the mutant 3'UTR fragment by point inactivating the miR-26b-binding sequence with site-directed mutation technique,and constructed the ROCK1 3'UTR wild-type and mutant dual luciferase reporter plasmids.The above reporter plasmids and miR-26 b mimic were simultaneously transferred into MSCs.By microplate reader assay,we found that the expression of the wild-type reporter gene was reduced,but the expression of mutant reporter gene was not changed.The results indicate that miR-26 b can bind to the wild-type 3'UTR,thereby inhibiting the expression of the reporter gene.Through the above experiments we confirm that miR-26 b can directly target ROCK1 and inhibit the expression of ROCK1.Subsequently,using the Boyden chamber device we investigated whether miR-26 b affects the migration of MSCs through ROCK1.The results showed that transfection of miR-26 b I,which inhibited the expression of endogenous miR-26 b and upregulated ROCK1,significantly inhibit the random migration and HGF-induced chemotactic migration of MSCs.Upon treatment with Y27632,a specific inhibitor of ROCK1,the random migration and chemotactic migration of MSCs were significantly increased both in control and miR-26 b I-treated group,indicating that the inhibition of ROCK1 activity by Y27632 can restore the inhibitory effect of miR-26 b I on MSC migration.In contrast,transfection of miR-26 b mimic,which upregulated miR-26 b and decreased ROCK1 expression,significantly promoted the random migration and chemotactic migration of MSCs.Infection of Ad-ROCK1,which restored the expression of ROCK1,significantly reduced the random migration and the chemotactic migration of MSCs,suggesting that upregulation of ROCK1 attenuated the miR-26b-promoted migration of MSCs.The above experimental results indicate that miR-26 b promotes the migration of MSCs by downregulating the expression of ROCK1 and inhibiting its activity.ROCK1 is a major downstream effector of Rho A.It has been reported that it can increase the phosphorylation of myosin light chain(MLC)on Ser19,leading to the formation of stable actin filaments and promoting their contraction.Moreover,ROCK1 can also promote the phosphorylation of Cofilin on Ser3 and regulate the cytoskeleton rearrangement.In addition,some studies have shown that ROCK1 can activate the phosphatase activity of PTEN,leading to the inhibition of Akt signaling pathway.In order to investigate which downstream molecules of ROCK1 regulate MSCs migration,we first detected the phosphorylation levels of Akt,Cofilin,and MLC after changing the expression of miR-26 b by Western blot.The results showed that when miR-26 b was upregulated,Akt signaling pathway was activated,but the phosphorylation levels of Cofilin and MLC were both decreased;when miR-26 b was downregulated,Akt signaling pathway was inhibited,but the phosphorylation levels of Cofilin and MLC were increased.These results indicate that miR-26 b can activate the Akt signaling pathway,which is consistent with our previous reports,but inhibit the phosphorylation of Cofilin and MLC.Further,we investigated whether ROCK1 is involved in the above changes.The results showed that overexpression of miR-26 b and ROCK1 at the same time can inhibit the activation of Akt signaling pathway,but restore the phosphorylation level of Cofilin and MLC,while inhibition of miR-26 b and ROCK1 at the same time can restore the activation of Akt signaling pathway,but inhibit the phosphorylation of Cofilin and MLC.The above results indicate that miR-26 b affects the activation of Akt signaling pathway and the phosphorylation of Cofilin and MLC by targeting ROCK1.The establishment of cell polarity is an important process for cell migration.Studies have pointed out that as a kinase,ROCK1 can change cell polarity and thus affect migration.We use phalloidin to show microfilaments by immunofluorescent staining,and determine cell area,perimeter,and the ratio of major axis to minor axis using Image J software to analyze cell spreading and polarity.The results showed that the area and perimeter of MSCs became smaller when miR-26 b was upregulated,and the cells showed a significant polar morphology.In order to study whether this phenomenon is related to ROCK1,we over-expressed miR-26 b and ROCK1 at the same time.It was found that both the cell area and the perimeter became larger,but the polar morphology disappeared when both miR-26 b and ROCK1 were highly expressed.The above results indicate that by targeting of ROCK1,miR-26 b decreases the spreading,promotes polarity formation,and promotes the migration of MSCs.Myosin is the main motor molecule that drives the contraction of cells.Actin filaments interact with myosins to form actomyosin fibers with contraction that regulate various biological processes,including cytokinesis,adhesion,and migration.The activated form of myosin is produced by the phosphorylation of the light chain,and the phosphorylation of MLC regulates the morphological changes of cells during adhesion,spreading and migration.We induced directional migration of MSCs overexpressing miR-26 b through wound healing,and detected the level,distribution and colocalization of phosphorylated MLC(p-MLC)with microfilaments by immunofluorescence staining.It was found that in MSCs overexpressing miR-26 b,the level of p-MLC was lower,which is consistent with the results of Western blot,and p-MLC accumulates at the leading edge of the cell membrane protrusions,whereas in the control cells p-MLC are mainly located in the intracellular stress fibers,but not in membrane protrusions or edges.These observations indicate that upregulation of miR-26 b leads to the enrichment of p-MLC at the MSCs' edges.In summary,the results of this study indicate that miR-26 b targets ROCK1,reduces the phosphorylation of Cofilin and MLC,promotes the distribution of p-MLC in the leading edge of migrating cell membranes,activates Akt signaling pathway,and affects the spreading and cell polarity of MSCs,thereby promoting the migration of MSCs.The results of this study further elucidated the molecular mechanism of MSC migration regulated by miR-26 b,providing theoretical and experimental basis for the clinical transplantation of MSCs.