| Objective:To analyze the relationship between Syndecan-1(SDC1) expression and tumor grades or prognosis in glioma; to determine the expression level of SDC1 in different human glioma cell lines and investigate the effect that SDC1 knockdown has on the proliferation and invasion of A172 and U87 cells and its related mechanisms; to confirm the effect of SDC1 knockdown in subcutaneous xenografts of U87 glioblastoma cell.Methods:1. Based on two publicly available databases: The Cancer Genome Atlas(TCGA) and the National Center for Biotechnology Information Gene Expression Omnibus(NCBI-GEO), the gene expression of SDC1 and tumor grades or prognosis were analyzed using the survival R package.2. The expression of SDC1 in glioma cells was analyzed through quantitative Real-time PCR(q RT-PCR) and Western Blot. Two stable SDC1-silencing cell lines A172 and U87 were constructed, which were named as sh SDC1 group; the cell lines transfected with scramble sh RNA were Scramble group; the untransfected cell lines were control group;3. Cell proliferation, invasion and immigration abilities were measured by MTT assay, trypan blue exclusion assay, flow cytometry and Transwell assays; q RT-PCR and Western Blot were performed to detect the expression of SDC1 and related proteins;4. Potential mechanisms in which SDC1 silencing inhibited the proliferation and invasion of glioma cells were investigated by Western Blot;5. Xenograft in nude mice was used to confirm the effect of SDC1 knockdown.Results:1.TCGA and NCBI-GEO show that high transcription level of SDC1 is correlated with advanced tumor grade(P<0.0001); Glioma patients with SDC1 expression level above two-third showed a great decrease in survival with regard to those with SDC1 expression level below one-third(P =0).2.q RT-PCR and Western blot showed a robust level of SDC1 in U87 and A172 cells and a low level of SDC1 in U251 and SHG-44 cells(P<0.05); The stable SDC1-silencing cells of A172 and U87 are established successfully.3. Compared with Control and Scramble groups, MTT assay, growth curve and colony formation assays showed that the proliferation of A172 and U87 cells in sh SDC1 group was significantly suppressed(P <0.05);flow cytometer indicated that the proliferation indices(S+G2/G1+S+G2) ofsh SDC1 group(30.84% ± 3.41% and 35.64% ± 5.24%) decreased significantly in comparison with Control group(47.76% ± 0.39% and47.20% ± 0.92%) and Scramble group(50.02% ± 3.39% and 45.69% ±1.44%) in A172 and U87 cells(P < 0.01), respectively. Transwell assay implied that cell migration(58.40 ± 5.24 vs 255.8 ± 16.09, 226.5±22.84 and 138.1 ± 3.21 vs 319.0 ± 10.91, 307.8 ± 8.83, P<0.01) and cell invasion(61.67 ± 16.26 vs 233.7 ± 17.24, 244.3 ± 28.15 and 840.7 ± 48.64 vs 1400± 53.84, 1416 ± 98.33, P<0.01) in sh SDC1 group were significantly decreased in A172 and U87 cells; q RT-PCR and Western blot showed a concomitant reduction of MMP-9 and PCNA in sh SDC1 group in A172 and U87 cells(P<0.01).4. Compared with Control and Scramble groups, the phosphorylation of Tyr 416 c-src and Tyr 397 FAK in sh SDC1 decreased by 1.61 and 2.21 times in A172 cell and by 1.78 and 1.5 times in U87 cells(P<0.05); the phosphorylation of Erk, p-38 mapk and Akt was suppressed 2.16, 3.55 and1.48 times in A172 and 2.07, 2.80 and 1.71 times in U87(P<0.05);5. Subcutaneous xenografts assay showed that the tumor volumes in sh SDC1 group and Scramble/Control group were 830 ± 500 mm3 and 4800± 1530 mm3(P<0.05), respectively; the mean tumor weight of sh SDC1 group was evidently reduced with respect to Scramble/Control group(0.89 ± 0.34 g vs 2.30 ± 1.05 g, P<0.05); no significant alterations in body weight were recorded between the two mice groups; immunohistochemicalstaining demonstrated that sh SDC1 group retained a lower level of SDC1 expression and decreased microvessel density compared with Scramble/Control group.Conclusions:1. Elevated expression of SDC1 in glioma is closely related with tumor grade and poor prognosis; SDC1 knockdown depressed glioma cell proliferation and invasion in vitro and in vivo.2. SDC1 knockdown may suppress the proliferation and invasion of glioma though impeding integrin αvβ3/αvβ5 by deregulation of c-Src/FAK signaling complexes. These findings may reveal a novel therapeutic target in this devastating disease. |
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