The Role Of Notch-1 Gene In Glioma And Its Effect On Akt-mTOR Signaling

Notch signaling system plays an important role in the tumorigenesis and developments. Previous studies in our laboratory have found that notch-1 gene expression was significantly higher and involved in the glioma-genesis. However, which kind of the biological behavior of glioma is regulated by Notch-1 and the mechanism is still not clear. In this study, we constructed lentivirus to express Notch-1 intracellular domain NICD (active form of Notch-1), and in the previous works we have build notch-1 gene RNA interference lentivirus. We used the two lentivirus to infection human glioma U251 cells, observed the effects of Notch-1 on glioma cell proliferation, apoptosis, invasion, angiogenesis and researched the mechanism.The research including four parts:Part I: cloning of notch-1 gene intracellular domain NICD and lentiviral packagingUsing a long fragment cloning method, we extracted the mRNA from human U251 glioma cells and then reverse transcribed it into cDNA which contain notch-1 gene intracellular domain NICD, amplificated it by polymerase chain reaction (PCR) and linked with TA cloning vector pCR-XL-TOPO. After sequenced, the correct sequence of NICD was digested and recombined with pNL-IRES2-EGFP lentiviral plasmid. The lentivirus were generated from 293T cells which co-transfection of pNL-NICD-IRES2-EGFP plasmid, packaging plasmids Helper and VSVG. Through ultrafiltration and centrifuge method, we got 2.00E+8 TU/ml of high-titer lentivirus. U251 glioma cells were infected with the lentivirus and transfection effeciency were observed by fluorescence microscope. The expression of NICD mRNA and protein were analyzed by RT-PCR and Western-blot. The results showed an increase about 1.6 times of mRNA and 1.4 times of protein in infected NICD-lentiviral U251 glioma cells compared with control. All these proved that we have succeed in cloning the segment of NICD and expressing it effectently in U251 glioma cells.Part II: Effects of Notch-1 on glioma proliferation, cell cycle, apoptosis and their mechanismsU251 glioma cells were infected with Notch-1 RNA interference or over-expression NICD lentivirus. The cell proliferation was assayed with MTT, cell cycle was determined by flow cytometry, apoptosis was checked by Annexin-V labeling and AO/EB staining, The expression of cell cycle-related proteins cyclin D1, CDK-4, P27 and apoptosis-related proteins Bcl-2, MCL-1, PARP-1, caspase-3, caspase-9 were examined by Western-blot. It was found that down-regulation of Notch-1 expression inhibited U251 glioma cell growth, cells were blocked in G1-phase (P< 0.05) and S-phase cells were decreased (P<0.05), apoptotic cells were increased, over-expression of Notch-1 have the contrary results. Down-regulation of Notch-1 expression inhibited cyclin D1, CDK-4, Bcl-2, MCL-1, PARP-1 and increased P27, caspase-3, caspase-9 protein expression in U251 glioma cells, whereas we got the contrary results in U251 glioma cells over-expressed NICD.Part III: Effects of Notch-1 on glioma cell invasion, angiogenesis and their mechanismsThe effects of Notch-1 gene on U251 glioma cell invasion were examined with matrigel coated transwell chambers, angiogenesis of tumor cell supernatant were measured through endothelial cell tube formation examine. The invasion-related protein MMP-2, MMP-3 and MMP-7 were analyzed by Western-blot, VEGF expression was detected with RT-PCR and ELASA. Results showed that down-regulation of Notch-1 expression inhibited U251 glioma cells invasion and MMP-2, MMP-3 expression, over-expression of NICD promotes U251 glioma cells invasion and invasion-related protein MMP-2 and MMP-7 expression, but there is no change in angiogenesis and VEGF expression. However, Results indicated VEGFR-2 expression was decreased in Notch-1 down-regulated U87 glioma cells. Part IV: Effects of Notch-1 on Akt-mTOR signaling pathwayWe observed the effects of notch-1 gene down-regulation and over-expression on the T-Akt, p-Akt, p-mTOR, p-70S6K and p-4EBP1 in U251 glioma cells using Western-blot analysis, which are the major components of Akt-mTOR signaling. Results showed that down-regulation of Notch-1 expression inhibited p-Akt, p-mTOR and p-70S6K expression. Whereas we got the contrary results in U251 glioma cells over-expressed NICD.ConclusionDown-regulation of Notch-1 expression inhibites U251 glioma cell proliferation, results in cell cycle arrest, promotes cell apoptosis and reduces tumor cell invasion. The expression of proliferation accelerating proteins cyclin D1, CDK-4 and anti-apoptosis protein Bcl-2, MCL-1, PARP-1 were inhibited, while the expression of proliferation inhibiting protein P27 and pro-apoptosis proteins caspase-3, caspase-9 were up-regulated. The Akt-mTOR pathway composition including p-Akt, p-mTOR, p-70S6K were inhibited. So Notch-1 probably promotes glioma cell survival, proliferation and invasion through regulating Akt-mTOR signaling and its downstream targets.