The Effects Of Hypoxia On The Proliferation Of Glioma Stem Cell And Its Mechanisms

Background:Gliomas are the most common primary brain tumors and confer a grave prognosis. Standard therapies, such as chemotherapy, surgery, and radiation, have had limited success in treating patients with high-grade gliomas. Despite the continuous development of new clinical therapies, the prognosis and survival of glioma patients remain dismal. It is hoped that a greater understanding of the molecular pathways involved in glioma cell proliferation and survival will lead to more effective targeted therapies.Recent researches highlight the importance of cancer-initiating cells in the malignancy of gliomas. These cells have been referred to as glioma stem cells, as they share similarities to normal neural stem cells in the brain. There is increasing evidence that malignant gliomas arise from and contain these minority tumor cells with stem cell-like properties. This subpopulation of tumor cells with the potential for self-renewal and multi-lineage differentiation that recapitulates the phenotype of the original glioma, plays an important role in glioma initiation, growth, and recurrence. Glioma stem cells seem the potential target of glioma therapy in future.Hypoxic regions are common in malignant tumors such as glioma, and are thought to play an important role in several facets of tumor pathobiology. Hypoxia has been associated with resistance to radiation and chemotherapies in tomor, as well as tumor invasion and poor patient survival. One well-characterized mechanism of the cellular response to reduced oxygen availability involves the enhanced expression of the hypoxia-inducible factor-la (HIF-1α). The Notch pathway, a highly conserved signaling network, is critical for a series of processes, including cell fate specification, differentiation, proliferation, and survival, and especially maintenance of neural stem cells. Dysregulated Notch signaling has been implicated in tumorigenesis.Objectives:(1) By testing the CD133+phenotype, cell vitality and gene expression of HIF-1α of glioma spheres cultured under normoxic and hypoxia microenvironment, we explored the impact of hypoxia on the proliferation of GCSs.(2) By testing the CD133+phenotype, cell vitality of glioma spheres and HIF-1α knockdown glioma spheres under hypoxia microenvironment, we validated the role of HIF-la on the proliferation of GCS cultured under hypoxia microenvironment. Also, by testing the transcription and expression of Notchl signal pathway relative genes, we investigate whether hypoxia, especially HIF-1α modulate Notch signaling to impact the proliferation of GCSs.Methods:(1) Glioma spheres were cultured in normoxic and hypoxia microenvironment for24and48h. Then CD133+phenotype was measured with flow cytometry, cell vitality was measured with CCK8method, mRNA expression of HIF-1α was measured with Real Time-qPCR and protein expression of HIF-1α was measured with Western Blot.(2) We constructed HIF-1α knockdown glioma spheres by silence RNA, then the two glioma spheres were cultured in hypoxia microenvironment for24and48h. CD133+phenotype was measured with flow cytometry, cell vitality was measured with CCK8method. Glioma spheres were cultured in normoxic and hypoxia microenvironment for24h, HIF-1α knockdown glioma spheres were cultured in hypoxia microenvironment for24h. Then cell cycle was evaluated by flow cytometry. The mRNA and protein expression of Notchl signal pathway relative genes were measured with Real Time-qPCR and Western Blot, respectively.Results:(1) Compared to normoxic group, CD133+phenotype, cell vitality and mRNA expression of HIF-1α were significantly elevated in glioma spheres cultured under hypoxia microenvironment.(2) Compared to hypoxia group, CD133+phenotype and cell vitality were significantly reduced in HIF-1α knockdown glioma spheres.(3) Compared to normoxic group, the percentage of G1cell of hypoxia group was significantly reduced, and the percentage of S and G2cell were significantly elevated. Compared to hypoxia group, the percentage of G1cell of HIF-1α knockdown glioma spheres was significantly reduced, and the percentage of G2cell were significantly elevated.(4) Compared to normoxic group, the mRNA and protein expression of Notchl signal pathway relative genes (Notch-1, jaggad-1, HES-1) were significantly elevated in glioma spheres cultured under hypoxia microenvironment. Compared to hypoxia group, the mRNA and protein expression of Notchl signal pathway relative genes were significantly reduced.Conclusions:(1) Hypoxia could promote the proliferation and cell vitality of GCSs and the protein expression of HIF-1α. The silence of HIF-1α could reduce the proliferation and cell vitality of GCSs. It suggested that HIF-1α may play an important role in this process.(2) Hypoxia could induce GCSs enter S phase, and promote the proliferation of GCSs. The silence of HIF-1α induced GCSs cycling arrest at G2/M phases and reduced the proliferation of GCSs.(3) Hypoxia and HIF-1α modulate Notch signaling to promote the proliferation of GCSs. The silence of HIF-1α inhabit the Notch signaling, therefore, the proliferation of GCSs was inhabited either.