Roles Of SPOCK1 In Human Glioma Cells And Its Molecular Mechanism Research In Vitro

Glioma is the most common malignancy in central nervous system, which is more aggressive and progressive. Because of the poor prognosis and high fatality, glioma severely threatens patients life and health. The malignant transformation of neurogliocyte and neurone is a complicated process, involving a large amount of oncogenes and tumor suppressor genes. Increasing evidence has demonstrated that SPOCK1 as an oncogene promoted various cancer cells proliferation and invasion. In our present study, we investigated the effect of SPOCK1 on glioma cells proliferation, migration and invasion, which provide a potential therapeutic target as well as a prognostic marker for glioma.SPOCK1, an oncogene, is frequently over-expressed in various cancer tissues. Increasing evidence has demonstrated that SPOCK1 had played important roles in proliferation, migration and invasion of tumor cells. Study by Miao et al. showed that SPOCK1 as a target gene of TGF-β1 could regulate lung cancer cells epithelial-mesenchymal transition and silencing of SPOCK1 obviously inhibited the proliferation and invasion of lung cancer cells. Previous study also found that SPOCK1, upregulated by CHD1 L, promoted proliferation and invasion of hepatoma carcinoma cells. SPOCK1 could promote proliferation and metastasis of gallbladder cancer cells trough the PI3K/AKT signaling pathway. The expressions of SPOCK1 in malignant glioma and pilocytic astrocytoma exhibited significant differences, which indicated that SPOCK1 had important effect on genesis and progression in glioma. However, the effect of SPOCK1 on proliferation and invasion of glioma cells and the underlying mechanisms are far from clear. In this study, we investigated the effect of SPOCK1 on the proliferation, apoptosis, migration and invasion through over-expressing exogenous and RNA-interfered endogenous SPOCK1 expression in glioma cells.this study through the following measures to discuss. Expression of the SPOCK1 gene in glioma cellsTo investigate the role of SPOCK1 in glioma cells, we chose U87 MG cells with higher SPOCK1 expression level for stable transfection with sh RNA vector toward SPOCK1 and U251 cells, in which SPOCK1 is infrequently expressed, for stable transfection with SPOCK1 expression vector. The expression levels of SPOCK1 was measured by real-time PCR and Western blot analysis. An efficient silencing of SPOCK1 protein and m RNA expression was shown in U87 MG cells transfected with the SPOCK1 sh RNA compared with negative control group. An obvious high level of SPOCK1 protein and m RNA expression was apparent in U251 cells transfected with SPOCK1 expression vector. Effect of SPOCK1 on glioma cells proliferationThe effect of SPOCK1 on the proliferation of glioma cells were measured by CCK8 assay. Silencing of SPOCK1 significantly inhibited the proliferation of U87 MG cells. While overexpression of SPOCK1 could promote the proliferation of U251 cells. Additionally, the ability of glioma cells to form colonies was also determined. The result showed that silencing of SPOCK1 significantly decreased the number of colonies formed by U87 MG cells. Overexpression of SPOCK1 did just the opposite, increasing colony formation in U251 cells. Moreover, the cell cycle progression was detected by flow cytometric analysis. The results demonstrated that silencing of SPOCK1 resulted in a larger fraction of the population in the G1 phase and a significant decrease in the proportion in G2/M phase. Overexpression of SPOCK1 reduced the number of cells arrested in G1 phase significantly. Since PCNA is an index for evaluating the ability of cell proliferation, we subsequently detected the expression of PCNA by immunofluorescence assay. As shown in Fig. 3, knockdown of SPOCK1 significantly downregulated the expression of PCNA in U87 MG cells. On the contrary, overexpression of SPOCK1 upregulated the expression of PCNA in U251 cell. The above findings provide evidence that SPOCK1 as an oncogene may promote glioma cells growth. Effect of SPOCK1 on glioma cells apoptosisTo further explore the mechanism of SPOCK1 in tumor growth, we focus on the role of SPOCK1 in cell apoptosis. As assessed by annexin V-FITC and PI staining, knockdown of SPOCK1 induced obvious apoptosis in U87 MG cells. While the apoptosis was inhibited by SPOCK1 overexpression in U251 cells. Moreover, the nuclear morphological changes in the apoptotic cells were revealed by the Hoechst 33342 staining. Silencing of SPOCK1 resulted in more bright chromatin condensation and nuclear fragmentation of the nuclei, which was significantly suppressed by SPOCK1 overexpression. To further confirm the effect of SPOCK1 on apoptosis, a number of apoptosis related proteins were determined. The results showed that knockdown of SPOCK1 induced increase in expressions of Bax, cleaved PARP and cleaved caspase-3 and decreased expression of Bcl-2 in U87 MG cells. The expression changes of these apoptosis related proteins were inverted when SPOCK1 was overexpressed in U251 cells. These data indicated that SPOCK1 promotes glioma cell growth by reducing cell apoptosis.Effect of SPOCK1 on glioma cells migration and invasionCell migration capacity was assessed by wound healing assay. knockdown of SPOCK1 obviously inhibited the migration capacity of U87 MG cells. When SPOCK1 was overexpressed in U251 cells, the migration capacity was increased significantly. Moreover, the invasive capacity was assessed by transwell assay. The results were similar to the changes in migration ability that was inhibited by SPOCK1 silencing and promoted by SPOCK1 overexpression. To further confirm the effect of SPOCK1 on metastasis, the activity and expression of MMP9 and MMP2 was determined by gelatin zymography and western blot. The activity of MMP9 and MMP2 was restrained by SPOCK1 silencing in U87 MG cells. While overexpression of SPOCK1 could promote the activity of MMP9 and MMP2. The changes of MMP9 and MMP2 expression were similar to those of MMP9 and MMP2 activity. Effect of SPOCK1 on PI3K/AKT and Wnt/β-catenin signaling pathwaysSince PI3K/AKT and Wnt/β-catenin signaling pathways are associated intimately with proliferation and metastasis of cancer cells, the effect of SPOCK1 on PI3K/AKT and Wnt/β-catenin signaling pathways was evaluated. Knockdown of SPOCK1 significantly suppressed the protein levels of p-PI3 K, p-AKT, Wnt and β-catenin in U87 MG cells, which could be reversed when SPOCK1 was overexpressed in U251 cells. Moreover, the downstream targets of Wnt/β-catenin were also assessed. The results indicated that the protein levels of c-MYC and Cyclin D1 were decreased by SPOCK1 silencing, which were increased obviously when SPOCK1 was overexpressed.Our results suggest that SPOCK1 may serve as an oncogene in glioma. Overexpression of SPOCK1 promotes glioma cells proliferation, migration and invasion via PI3K/AKT and Wnt/β-catenin signaling pathways, which could be reversed by SPOCK1 silencing. These observations confirm that SPOCK1 may serve as both a treatment target and prognostic indicator for patients with glioma.