| Malignant glioma is a highly invasive brain tumor resistant to the conventional therapies, such as surgical operation, chemotherapy and radiotherapy. Secreted protein acidic and rich in cysteine (SPARC) has been shown to facilitate glioma invasion. However, the effects of SPARC on cell growth, invasion and radiosensitivity have not been adequately elucidated. In this study, I constructed a plasmid expressing small interfering RNA (siRNA) against SPARC, evaluated the effect of SPARCsiRNA on SPARC expression and then assessed its effects on cell growth, invasion and radiosensitivity in U-87MG cells and the potential mechanisms.With a siRNA expression plasmid vector targeting SPARC, I obtained the stably transfected cells in which the expression of SPARC was downregulated successfully. Cell growth curves and colony formation assay suggested that the introduction of SPARCsiRNA resulted in an increase of cell growth and colony formation. Wound healing assay indicated that the invasive capacity of U-87MG cells was deceased by SPARCsiRNA. It was shown that the knockdown of SPARC expression was capable of promoting the cell cycle progression from G1 to S phase. However, no difference was found in apoptosis. Cells were then irradiated with 60Co-γ-rays and analyzed by several methods, including clonogenic assay, 3H-TdR incorporation assay, flow cytometry, comet assay and Western blotting. Clonogenic assay showed that a downregulation of SPARC expression enhanced cell survival caused by radiation. IR-induced inhibition of DNA synthesis was attenuated by SPARCsiRNA. Flow cytometry analysis indicated that SPARCsiRNA decreased cell apoptosis responding to irradiation. Furthermore, cell cycle analysis showed the downregulation of SPARC enhanced G2 accumulation after irradiation. Comet assay revealed SPARCsiRNA promoted the repair of radiation-induced DNA damage. A molecular analysis of signal mediators indicated that the decrease of p-c-Raf and accumulation of p-GSK-3β, p-AKT and p-PDK1 may be associated with the growth promotion by SPARCsiRNA. And the level of Akt phosphorylation was increased in irradiated U-87MG/SPARCsiRNA cells, which was suppressed by a PI3K inhibitor (LY294002). To determine the role of SPARC in vivo, U-87MG/SPARCsiRNA, U-87MG/controlsiRNA and U-87MG cells were injected stereotactically into the brains of athymic nude mice. MRI demonstrated a significant increase in tumor volume of U-87MG/SPARCsiRNA mice. After irradiation, the rate of tumor inhibition was decreased by SPARCsiRNA. The examination also revealed that tumor invasion was reduced in U-87MG/SPARCsiRNA group. Immunostaining revealed that SPARC knockdown increased the proportions of PCNA and CD31 positive cells. Western blot analysis showed that the level of Akt phosphorylation was also increased by SPARCsiRNA in vivo. Tissue samples of 16 glioma patients were collected and further examined by immunostaining to evaluate the expression level of SPARC. The results showed that a highest level of SPARC was found in the cases of I-II tumor grade, up to 53.46%. In 60% female patients, SPARC-positive cells were more than 10% of total cells, while in all of the male patients, SPARC-positive cells were less than 10%.Our results suggest that inhibition of SPARC expression may promote the growth, inhibit the invasion and diminish the radiosensitivity of human glioma both in vitro and in vivo. The acceleration of cell cycle progression from the G1 to S phase might result in the growth promotion by SPARCsiRNA. The reduced cell apoptosis, enhanced G2 accumulation and increased DNA repair after exposure to irradiation may account for the decreased radiosensitivity by SPARCsiRNA. The activation of PI3K/AKT pathway and the involvement of GSK-3βand c-Raf may be the mechanisms for the growth promotion and decreased radiosensitivity. |
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