Combination Of PTEN Reconstruction And VEGF Knockdown Offered A Potential Gene Therapeutic Strategy For Malignant Gliomas.

Glioblastoma (GBM) is a highly malignant tumor with poor prognosis. High expression of vascular endothelial growth factor (VEGF) and the depletion of PTEN are two of the most important hallmarks of GBM. To date, combined therapies are becoming a promising strategy owning to its potential ability of overcoming tumor drug resistance produced in monotherapy via inhibiting different signaling pathways. So in present research, we want to determine whether combined gene therapy of wild-type PTEN reconstruction and VEGF siRNA is a more effective approach for inhibition of tumor growth and tumorigenicity of PTEN-null glioblastoma.1) The results of recombination vectors and the stable expression cell lines. siRNA sequences were synthesized and inserted between the BamHI and Hindâ…¢restriction sites of pRNATU6.1/Neo for shRNA expression. PTEN cDNA sequence was cloned into tetracycline-inducible retroviral expression vector pRetro-on. The recombinant construct was confirmed by both restriction enzyme analysis and sequencing. Stable PTEN inducible U251 cell line was selected with lug/mL puromycin and induced by lug/mL doxycycline. Double stable expression of PTEN and shRNA were selected by 600ug/mL G418.2) The efficiency of VEGF shRNA construct and the inducd expression of PTEN. The mRNA level of VEGF was tested by real-time PCR at 48h post-transfection.. Protein expression of VEGF in the cell culture supernatant were determined by ELISA. We get a marked knockdown effect:58.5%and a significant reduction of VEGF secretion level. At 48h post-transfection and doxycycline treatment, the results of western blot showed that PTEN was successively induced.3) The results of cell proliferation, cell cycle and cell apoptosis. Cell proliferation was measured by MTT assay and phospho-Akt protein expression was detected by Western blot. Cell cycle was analyzed using flow cytometry and cell early apoptosis was detected by annexin V-PE staining. PTEN restoration could affect proliferation, arrest cell cycle at G0/G1 stage and promote apoptosis of U251 glioblastoma cells via inhibition of PI3K/AKT signaling pathway in vitro.4) The results of colony formation ability and cell migration.Colony formation ability was analysed by Anchorage-dependent and independent conony formation assay. Cell migration ability was measured using a wound-healing assay. In spite of the fact that transient transfection of VEGF shRNA failed to affect the growth and cell cycle progression of U251 cells, the VEGF siRNA seemed to efficiently inhibit the anchorage-dependent and-independent colony formation ability and wound-healing migration ability of U251 cells with VEGF siRNA stable expression. Furthermore, combined treatment of PTEN and VEGF siRNA had the best suppression effect.5) Subcutaneous tumor model of nude mice was constructed to evaluate the inhibition ability of PTEN and VEGF siRNA on growth of U251 cells. Similar to the results in vitro, null-mice xenograft study showed that the restoration of wilt-type PTEN or the expression of VEGF siRNA could significantly inhibit the growth of U251 GBM respectively and no obvious tumors were observed in the combined treatment group.In a word, the combination of PTEN expression and VEGF knockdown demonstrates an effective gene therapeutic strategy for malignant gliomas.