Expression Of Notch 1 In Human Gliomas And Construction Of The Eukaryotic Expression Vector Of Human Notch 1 Intracellular Domain

Notch signaling is an evolutionarily highly conserved pathway, which plays a fundamental role during the embryonic development of vertebrates and invertebrates. Signals exchanged between neighboring cells through the Notch receptor can amplify and consolidate molecular differences, which eventually dictate cell fates.Notch ligands and receptors are type I membrane proteins. To date, four Notch receptors (Notch 1~4) have been identified in humans, with five corresponding ligands including Delta-like-1, Delta-like-3, Delta-like-4, Jagged-1, and Jagged-2. Activation of Notch signaling requires binding of an adjacent cell's ligands Delta-like or Jagged. Upon ligand binding, the intracellular portion of the Notch receptor is cleaved and enters the nucleus, where it influences the expression of numerous transcription factors. Thus, Notch signaling can positively or negatively influence proliferation, differentiation, and apoptosis.Notch signaling has long been shown to have a profound effect on many aspects of nervous system development, including neuronal differentiation and glial determination. Notch signaling can inhibit neuronal differentiation and directly promote the differentiation of astrocyte. However, oligodendrocyte precursors derived from glial progenitors fail to differentiate into mature oligodendrocytes in the presence of active Notch signaling.The importance of Notch signaling in regulating cell fate decisions in the vertebrate nervous system suggests its potential for aberrant regulation in glioma cells. Purow et al. showed that Notch-1 and its ligands, Deltalike-1 and Jagged-1, are overexpressed in many glioma cell lines and primary human gliomas and that down-regulation of Notch 1, Delta-like-1, or Jagged-1 by RNA interference induces apoptosis and inhibits proliferation in multiple glioma cell lines. Paradoxically, Somasundaram et al. showed that inhibition of Notch signaling may be an important early event in the development of gliomas.Is it possible that activation of Notch signaling can actually be bad for the growth of gliomas? Resolution of the Notch signaling paradox may come from better understanding the mechanism of Notch signaling in gliomas genesis and progression. For this, the current study was performed from two aspects as follows.I. Expression of Noch 1 mRNA in glioma cells and its significance: Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of Notch 1 mRNA in four human glioma cell lines (U251, U87, BT325, SHG-44). 159 cases of human brain glioma tissue and 15 cases of human normal brain tissue were investigated by immunohistochemistry for the expression of Notch 1 protein. The results showed that, Notch 1 mRNA was expressed in four glioma cell lines. There was significant difference (P<0.05) respectively in the positive expression rate and immunoreactivity score (IRS) of Notch 1 protein between astrocytic tumors or oligodendroglial tumors and normal brain tissue, whereas no significant difference (P>0.05) between medulloblastoma and normal brain tissue. Among different pathological grades of astrocytic tumors, there was no significant difference (P>0.05) in the positive expression rate of Notch 1 protein, whereas there was significant difference (P<0.05) between IRS of Notch 1 protein in pilocytic astrocytoma (WHO grades I) or anaplastic astrocytoma (WHO grades III) and that in diffuse astrocytoma (WHO grades II) or glioblastoma (WHO grades IV). Among different pathological grades of oligodendroglial tumors, there was no significant difference (P>0.05) in the positive expression rate of Notch 1 protein, whereas there was significant difference (P<0.05) in IRS of Notch 1 protein between oligodendroglioma (WHO grades II) and anaplastic oligodendroglioma (WHO grades III). The results suggested that, Notch 1 protein expression was significantly higher in astrocytic tumors and oligodendroglial tumors than in normal brain tissue, and that Notch signaling may associate closely with tumorigenesis and development of gliomas.II. Construction of the eukaryotic expression vector of human Notch 1 intracellular domain: Total RNA was extracted from the U251 cells and reverse transcription-polymerase chain reaction (RT-PCR) was performed to obtain the cDNA fragment encoding NICD, which was inserted into the pIRES2-EGFP vector in-frame. And the new construct was confirmed by restriction enzyme digestion and DNA sequencing. HeLa cells were transfected with the pIRES2-EGFP-NICD vector and pIRES2-EGFP vector, respectively. The expression of NICD was detected by Western blotting. The results showed that, the cDNA fragment encoding NICD was cloned into the pIRES2-EGFP vector in-frame, and significant increase of NICD expression was detected in the HeLa cells 72 hours after transfection. The results suggested that, the eukaryotic expression vector pIRES2-EGFP-NICD was constructed successfully. This study laid a foundation for the research on the mechanism of Notch signaling in tumorigenesis and development of gliomas.