| Gliomas is the most frequent type of primary tumor of the central nervous system.Gliomas can be classified into classes Ⅰ-Ⅳ according to their histological characteristics.The incidence of malignant gliomas is particularly higher among the patients with glioma,and its ability to invade the surrounding brain.Although malignant gliomas comprise only small percentages of all adult tumors(4-5/100,000 adults/year).However,their highly invasive properties make them the fourth largest cause of cancer related deaths.This invasive behaviour precludes total resection of the tumour,leading to rapid recurrence and treatment failure.Whereas the molecular mechanisms of malignant glioma invasion are incompletely understood,there is growing evidences that cytoskeletal-matrix interactions contribute to glioma process.Nexilin is a novel actin filament-binding protein localized at cell-matrix adherens junction,isolated from rat brain.Until recently,limited data existed on the role of Nexilin in tumor.Nelin is a homologous gene of Nexilin and mediates cell motility.HGF upregulated Nexilin expression in neonatal cardiomyocytes.In view of the evidence,we hypothesized that the dysregulation of Nexilin occurs relatively in the glioma process and may be promising for cancer therapy.Objectives:1.To investigate whether Nexilin expression is changed in glioma,arid correlated with clinicopathological parameters.2.To construct recombinant plasmids that expressed miRNA targeting the Nexilin,based on the plasmid pcDNA6.2-GW/EmGFP-miR.3.To investigate the effects of Nexilin on cell proliferation,adhesion,migration and invasion of human glioma by downregulation of Nexilin expression.4.To investigate the effects and mechanism of Nexilin on HGF-induced migration and invasion of human glioma.We will clarify the effects of Nexilin knockdown by shRNA on HGF-induced migration and invasion of glioma,and associated signaling pathways in vitro.Methods:1.Expression of Nexilin in glioma tissue:Nexilin expression was determined by immunostaining analysis using an anti-human Nexilin monoclonal antibody of a glioma TMA,as well as determined by western blot analysis of four paired glioma tissues and adjacent non-tumor tissues from the same patients.2.Contraction of recombinant plasmid containing miRNA targeting Nexilin gene:The human cDNA sequence encoding Nexilin gene(Gene ID:91624)was obtained from Genbank,based on which four pairs of miRNA oligo were designed and synthesized.After annealing into double-strain miRNA oligo,recombinant plasmid(Nexilin shRNA)was constructed using pcDNA6.2-GW/EmGFP-miR Vector.Transient transfections of shRNA against Nexilin were carried out using Lipofectamine 2000.Nexilin expression was determined by western blot analysis.3.The effects of Nexilin knockdown on proliferation,attachment,migration and invasion in glioma cells:Transient transfections of shRNA against Nexilin were carried out using Lipofectamine 2000.Cell proliferation was determined using the MTT assay.Cell migration and invasion was assessed by matrigel precoated Transwell inserts according to the manufacturer’s protocol.The cell attachment assay was performed in 24-well plates precoated with fibronectin.Plates were blocked by PBS containing 2.5 mg/ml bovine serum albumin for 2 hour at 37℃.Cells were then plated on coated culture plates and incubated for 1 hour.Unattached cells were removed by washing with serum-free medium at the end of the incubation period,andthe attached cells were determined using the MTT assay.4.The effects of Nexilin knockdown by shRNA on HGF-induced migration and invasion of glioma cells,and associated signaling pathways in vitro:Recombinant human HGF was dissolved in 0.1%BSA in PBS and stock solution was stored at-20℃.Specific depletion of Nexilin was accomplished using shRNA.Cell migration and invasion was determined using wound-healing and matrigel assays in the presence or absence of HGF for 12 hours,24 hours,and Erk、JNKand AKT as well as phosphorylation of Erk、JNKand AKT were determined by western blot analysis in the presence or absence of HGF for 30 min.Results:1.Expression of Nexilin in glioma tissue:Nexilin positive staining was observed in the all of normal brain tissue,tumor adjacent normal brain tissue and glioma tissue.Nexilin protein mainly localized in cytoplasm.The expression of Nexilin was significantly higher in glioma tissue than that in normal brain tissue and tumor adjacent normal brain tissue by immunohistochemical staining(P<0.001).Nexilin staining was increased in WHO stages Ⅱ,Ⅲ and Ⅳ compared with stages Ⅰ(P<0.001).The expression of Nexilin was increased in the glioblastoma than that of low grade astrocytoma and ependymoma.There is also no significant correlations between Nexilin expression with other clinicopathologic variables,including patient age and gender.To further confirm these results,a western blot assay was finished using four glioma tissues and paired non-tumor tissues.It was clear that the expression of Nexilin in glioma tissue was significantly higher as compared with the non-tumor tissues,which was consistent with the observations of immunohistochemical staining.In addition,western blot analyses showed that the expression of Nexilin was markedly higher in 4 analyzed glioma cell lines,including A172,U251,U87 and SH-SY5Y,as compared with the normal human astrocytes(NHA).Collectively,our results suggest that Nexilin is Overexpression in gliomas.2.The construction and identification of plasmids containing miRNA targeting Nexilin gene:Sequencing confirmed that the double-strain miRNA oligo Were exactly inserted into vectors,indicating that shRNA recombinant plasmids vectors targeting Nexilin gene were constructed successfully.Nexilin protein level in A172 cell transfected by shRNA-3 recombinant plasmid were significantly lower compared with negative plasmid or untransfected control,in 48 hour after transfection,which indicated that shRNA recombinant plasmid targeting Nexilin gene can inhibit Nexilin gene expression effectively.3.The effects of Nexilin knockdown on proliferation,attachment,migration and invasion in glioma cells:We transfected glioma A172 and U87 cells with shRNA recombinant plasmid targeting Nexilin and found that Nexilin was markedlydown-regulation in these cell lines compared with control cells.Subsequently,48 hour after transfection,cells were subjected to cell proliferation,attachment,migration and cell invasion assay.Our data indicated that the cell proliferation rates were decreased in Nexilin knockdown groups than control group in both A172 and U87 cells.We found that cell attachment ability was reduced by 38.03%and 21.38%in Nexilin knockdown A172 and U87 cells compared with the control cells.In cell migration assay,our results showed that A172 and U87 cells of Nexilin knockdown decreased the ability to migrate through Boyden chamber by 49.6%and 57.6%,respectively,compared with the control cells.In cell invasion assay,Nexilin elimination inhibits cell invasive ability of A172 and U87 cells in matrigel-coated Boyden chamber by 55.5%and 34.7%,respective.4.The effects of Nexilin knockdown by shRNA on HGF-induced migration and invasion of glioma cells,and associated signaling pathways in vitro:HGF upregulated Nexilin protein expression by 3.5 fold in U87 glioma cell after 24 hours.The HGF-promoted cell migration and invasion were inhibited when Nexilin was knocked down by shRNA in U251and U87 cells.Depletion of Nexilin suppressed the Erkl/2、JNK(45KD)and AKT(ser473)phosphorylation in U25 land U87 cells.Conclusions:1.Our results suggest that Nexilin is Overexpression in gliomas.2.Our data indicated that shRNA recombinant plasmids vectors targeting Nexilin gene were constructed successfully,and can inhibit Nexilin gene expression effectively.3.Nexilin elimination inhibits cell proliferation,attachment,migration and invasive ability of A172 and U87 cells.4.HGF upregulated Nexilin protein expression.Nexilin mediates HGF-induced migration and invasion of glioma cells.5.Nexilin knockdown by shRNA suppressed the Erk1/2、JNK(45KD)and AKT(ser473)phosphorylation. |
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