Study Of Neuregulin-1 Impacting Bone Marrow Mesenchymal Stem Cells Migration Through Regulation Of Snail Expression

Backgroud:Spinal cord injury(SCI)is a kind of disease with high disability and high mortality,we not only have been troubled in physical and mental health,but also have economy and society pressure.Cell transplantation and gene therapy,a hot research topic and direction,has become the the fastest growing science in progress to settle up these medical problems after 2000.Bone marrow mesenchymal stem cells(BMSC)are increasingly used for cell transplantation in spinal cord injury(SCI)and get more concentration since Friedenstein,et al confirmed they really exist in 1968.They may transform into neuron cells and improve structural and functional repair in spinal cord injury location.In addition,BMSC can secrete a wide array of cytokines including vascular endothelial growth factor(VEGF),hepatocyte growth factor(HGF),fibroblast growth factor(FGF),insulin-like growth factor(IGF),stem cell-derived factor(SDF)and so on to promote endogenous cells proliferation and growth.Neuregulins(NRGs)are a family of growth and differentiation factors that are related to epidermal growth factor(EGF).Their receptors,including Erb B1/EGF receptor,Erb B2/neu,Erb B3 and Erb B4,are the Erb B family of tyrosine kinase transmembrane receptors.NRGs reacting with Erb B3 or Erb B4,are involved in cell proliferation,migration,differentiation,growth,survival and apoptosis by emerging homoplasmon or heteroplasmon dimeride binding with Erb B1 or Erb B4 subsequently.Alternative splicing of the NRG1 gene results intranscripts encoding 3 major isoforms.Type I NRG1,also known as heregulin(HRG),a 44 k D glycoprotein,is expressed predominantly in the neural tissue?respiratory epithelium and endocardium.HRG can promote the survival of neuron,glial cells,epithelial cells,cardiomyocytes and other cell types binding with Erb B receptor.The epithelial-mesenchymal transition(EMT),originally defined as a morphological conversion occurring at specific stage of embryo development,is implicated in the conversion of early stage tumors into invasive malignancies.The well-established role of Snail in EMT during both embryogenesis and tumor progression reflects its possible participation in migration of stem cells.In spite of the implication of Snail in invasion/metastasis of cancer cells,little is known about its role and function in BMSC.In our research,we assumed Snail is involved in the migration of BMSC,and modified it with Snail expression plasmid vector.We demonstrated that expression of Snail increased the migration of BMSC and upregulated MMP-2 expression in vitro and enhance the phenomenon with exogenous Neuregulin-1.Forthurmore,we observed cells diffusion in injury location in Allen spinal cord injury model.Based on these findings,we believe the transplanted BMSC is easier to migrate to the injured location in microenvironment.Purpose:1?We investigate the effect of Neuregulin-1 on the expression of Snail of rat bone marrow mesenchymal stem cells(MSCs)in vitro,and determine the optimal concentration and time to promote Snail m RNA expression of bone marrow mesenchymal stem cells.2?We investigate the effect of Neuregulin-1 on migration of rat bone marrow mesenchymal stem cells(MSCs)and regulation of transcription factor Snail and matrix metalloproteinase-2(MMP-2)expression.3?We investigate if the rat bone marrow mesenchymal stem cells transfected with transcription factor Snail can promote the motor function recovery in animal models of spinal cord injury.Experimental Methods:Application of the relative quantitative PCR(qRT-PCR)method for detection of Neuregulin-1 on 0ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml concentration,respectively in rat bone marrow mesenchymal stem cells 24 hours,48 ??hours,72 hours after the rat bone marrow mesenchymal stem cells in the case of Snail m RNA expression.Snail over-expression plasmid(p Babe-puro-Snail)and negative control plasmid(p Babe-puro)were transfected into rat bone marrow mesenchymal stem cells(BMSC)with Lipofectamine 2000.MTT verify if Snail over-expression plasmid(p Babe-puro-Snail)and negative control plasmid(p Babe-puro)generated on rat BMSC proliferation.Application of Neuregulin-1 on the concentration of 40ng/ml 48 hours,we detect Snail m RNA,MMP-2 m RNA and the Snail,MMP-2protein expression with q RT-PCR and Western Blot respectively.Transwell cell migration assay detect the migration of BMSC with Neuregulin-1.36 SD rats,prepared spinal cord injury model,were divided into A,B,C,D,E,F groups randomly,group A application of rat bone marrow mesenchymal stem cells transfected with Snail and Neuregulin-1(BMSC-Sna+NRG),group B application of rat bone marrow mesenchymal stem cells transfected with Snail-NC and Neuregulin-1(BMSC-NC+ NRG),group C application of rat bone marrow mesenchymal stem cells and Neuregulin-1(BMSC+NRG),group D application of rat bone marrow mesenchymal stem cells transfected with Snail(BMSC-Sna),group E application of rat bone marrow mesenchymal stem cells transfected with Snail-NC(BMSC-NC),group F application of rat bone marrow mesenchymal stem cells(BMSC).We record the BBB function score of each group after SCI in 4 weeks,analyze the data with SPSS12.0.Observe the quantity and the area of transplanted cells in the local injured region in fluorescence microscope.Results:NRG1 significantly increases the m RNA expression level of Snail in a concentration-dependent manner.The effects of different concentrations of NRG1(0-80ng/ml)on the m RNA expression level of Snail were detected by RT-q PCR analysis at 24,48,and 72 h.As shown in Fig.2A,NRG1 significantly increased the m RNA expression of Snail in a concentration-dependent manner between 0 and 40 ng/ml,with a peak at 40ng/ml.At the dose of 80 ng/ml NRG1,the expression level of Snail was significantly lower than that at the dose of 40 ng/ml NRG1.The m RNA expression level of Snail was significantly higher at 48 h than that at either 24 or 72 h post-NRG1 treatment(P<0.05).These results suggested that NRG1 at a dose of 40 ng/ml for 48 h led to the most effective increase on the expression of Snail in BMSC,which was the optimal condition used in the following experiments.Effects of NRG1 on the m RNA and protein expression levels of Snail and MMP-2.A total of six groups were included in the study: Group A(BMSCs transfected with Snail overexpression plasmid plus NRG1;BMSC-Sna + NRG1),group B(BMSCs transfected with Snail negative expression plus NRG1;BMSC-NC + NRG1),group C(BMSCs plus NRG;BMSC + NRG1),group D(BMSCs transfected with Snail overexpression;BMSC-Sna),group E(BMSCs transfected with Snail negative expression;BMSC-NC)and group F(BMSCs).As shown in Fig.2B,the expression levels of Snail m RNA in groups A and D were significantly higher than those in groups B,C,E and F due to transfection with the Snail overexpression plasmid(P<0.05).There were significant differences between group A and group D(P<0.05).Furthermore,the m RNA expression levels of MMP-2 in groups A and D were significantly higher than those in groups B,C,E and F,which indicated that the overexpression of Snail stimulated the expression of MMP-2,and NRG1 further enhanced this positive effect of Snail(P<0.05).As shown in Fig.3,no significant differences in cellular viability were observed between groups D,E and F at different time points(P>0.05).This indicated that the cellular growth of BMSCs did not significantly alter due to transfection with the Snail overexpression plasmid.According to A-C,he results of the western blot analysis revealed that the expression levels of Snail in groups A and D were significantly higher than those of groups B,C,E and F(P<0.05).No significant differences were observed between groups A and D(P>0.05).Similarly,the expression levels of MMP-2 in groups A and D were significantly higher than those of groups B,C,E and F(P<0.05).Cell migration of BMSCs in vitro.As shown in A and B,the numbers of cells crossing the membrane in groups A and D were significantly higher than those in groups B and C(P<0.05).Additionally,there were significant differences between groups A and D in the numbers of cells that crossed the membrane(P<0.05).However,no cells appeared to cross the membrane in groups E and F.This suggested that NRG1 enhanced the promoting effect of Snail on BMSC migration.BBB scores and assessment of functional recovery.Two experts were invited to perform the BBB rating scale in a blinded-manner at the end of each week between weeks 0 and 4following cell transplantation.The BBB scores of group D were significantly higher than those of groups B,C,E and F between 1 and 4 weeks post-BMSC transplantation(P<0.05).The BBB score of group A was significantly higher than the scores of the other groups at different time points,including weeks 2,3 and 4(P<0.05).In addition,the distribution area of BMSCs in group A was significantly larger than those of the other groups and the distribution area of BMSCs in group D was significantly larger than those of groups E and F.Conclusion:Exposure of cells to Neuregulin-1 showed a concentration-dependent increased of snail expression,and mesenchymal stem cells incubated on 40ng/ml of Neuregulin-1 showed a maximum snail expression.Exogenous Neuregulin-1 can increase the expression of Snail,thus contributing to the expression of MMP-2,enhance the bone marrow mesenchymal stem cell migration.Neuregulin-1 can promote the transfection of Snail in the rat bone marrow mesenchymal stem cells to treat spinal cord injury recovery of motor function.