Osteopontin In Human Glioma Associated Preliminary Study Of Gene Therapy

Research background:Osteopontin (OPN), a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family of proteins, is a secreted multifunctional glycoprotein expressed at high levels in various tumors. It has been reported that expression level of OPN correlated with tumor grade and prognosis in patients with breast, stomach, prostate, liver, colon and lung cancers, and OPN has been identified as a biomarker for tumor progression in many human tumours. OPN stimulates a complex downstream signaling cascade by binding to theαvβ3 integrin and CD44 receptors and is associated with cell apoptosis, proliferation, migration and angiogenesis. Studies have shown that expression level of OPN correlated with the malignancy in human glioma cells. It is also demonstrated that overexpression of OPN protein in astrocytoma cells and suggested OPN was associated with astrocytoma progression and angiogenesis. Recent study showed that all three messages including OPN-a, OPN-b and OPN-c were present in breast cancer tissues and had different roles in human breast cancer progression, and increased OPN-c levels was also detected in metastatic HCC samples and a highly invasive HCC cell line (MHCC-97). Three types of splice variants of OPN in glioma cells have been isolated and sequenced by Saitoh et al in the year 1995. However, to date, there have been no reports which have analysed the expression pattern and functions of OPN splice variants in glioma cells.Research objection:1. The expression level of OPN in glioma tissues and the effects of OPN-silencing on the apoptosis and invasion of glioma cells.2. Expression pattern of OPN splice variants and its functions on cell apoptosis and invasion in glioma cells.Research Methods:Expression levels examination: we examined the expression profile of total OPN and three OPN splice variants in glioma tissues and glioma cells lines by real-time qPCR and RT-PCR. Function Study: OPN siRNA mediated by lentivirus was constructed to down-regulate the expression of OPN in U251 cells. And apoptosis and invasion of infected U251 cells were evaluated. To further investigate the biological function of OPN splice variants in the progression of gliomas, knock-down and regain of function experiments were employed. Expression vectors mediated by lentivirus for human OPN siRNA, OPN splice variants synonymous mutants and negative control vector were constructed via technical support from Shanghai GeneChem. OPN siRNA was designed to silence all three endogenous OPN splice variants using an Internet application system (Invitrogen, CA). And the expression of OPN in human tissues and cells were detected by real-time fluorescence quantitative PCR. The effects of the invasion and apoptosis of infected glioma cells were evaluated via transwell assay and Flow cytometric method.Research Results:1. The levels of OPN mRNA in the WHO-ⅢandⅣgrade glioma tissues were significantly higher than those in the WHO-Ⅱgrade glioma and normal brain tissues.2. OPN siRNA inhibited OPN expression, cell invasion and promoted apoptosis in U251 cells, moreover, Bcl-2, uPA, MMP-2 and MMP-9 expression were decreased by OPN siRNA, while Bax level was elevated.3. The average expression of OPN splice variants mRNA including OPN-a, -b and -c was significantly higher in high grade (grade III-IV) than low grade (grade I-II) gliomas by real-time qPCR. To confirm the expression pattern of OPN splice variants in human glioma cell lines, RT-PCR was carried out. The three bands of OPN splice variants were detectable in U251 and U87 cells, but scanty bands were seen in SHG44 and TJ905 cells.4. All three OPN splice variants show an inhibition of apoptosis induced by OPN siRNA in U251 and U87 cells in vitro and OPN-b elicited the strongest effect. Furthermore, OPN-a Mu and OPN-c Mu, but not OPN-b Mu, improve glioma cell invasion significantly.Research Conclusions:1. OPN siRNA can inhibit U251 cells invasion via the down-regulation of uPA, MMP-2 and MMP-9 levels, and promote apoptosis through induction of Bax expression and inhibition of Bcl-2 level. It suggests that OPN plays an important role in human glioma progression.2. In view of our evidences that splice variants of OPN have different roles in determining the fate of malignant glioma cells, we recommend that the difference among OPN splice variants could be a novel anticancer direction when developing new anticancer therapeutic approaches targeted to OPN splice variants in malignant gliomas.