NOTCH-1 Signaling Regulates Proliferation And Differentiation Of Human Brain Glioma Stem Cells

Glioma, as the most frequently malignant brain tumor in central nervous system (CNS), who is the most important problem in treating, due to its unlimited proliferation and invasive growth. Brain glioma stem cells are found in glioma, but they take up a small portion of tumor population,and are capable of self-renew, proliferation and differention. BGSCs are the "seeds" of glioma cells with any differented degree, play a key role in initiation, progression and recurrence of glioma. That is why we choose BGSCs as a cell model.NOTCH-1 signaling pathway was found to be correlated with human tumor formation in recent studies. It was detected in many tumors. Overexpression of NOTCH-1 may give rise to brain tumor in CNS. NOTCH-1 signaling may play a key role in the genesis of brain tumor, especially glioma. NOTCH-1 signaling is essential to maintenance and survival of many gliomas, medulloblastomas and the model system; NOTCH-1 signal may participate in ininitiation of glioma, and usually affect neural precursor cells or neural stem cells. NOTCH-1 signaling take effect through NOTCH-HES, and the expression of ( basic-helix-loop-helix,bHLH) is a reliable gene of cell reaction to the NOTCH-1signal. It may also control proliferation and survival of tumor through Cyclins, anti- apoptosis factor and cooperation with other signals, such as HH signaling.Since NOTCH-1 signaling pathway plays a key role in self-renew, proliferation and differentiation of NSCs or neural precursor cells, and ectopic activation of NOTCH-1 signal was detected in many gliomas (including medulloblastomas), as well as some other tumors and cell lines, we hypothesized that there may be ectopic activation of NOTCH-1 signal in BGSCs, seeds of glioma, which may play a key role in initiation and progression of glioma. But we must kown how did it happen? How did these changes decide the fate of BGSCs and control its proliferation and differentiation?In Part 1 of our research, BGSCs were sorted through immunomagnetic beads marking by CD133 and cultured in vitro, and character as a stem cell was identified by stem cell markers (CD133 and Nestin) and differentiated cell markers MAP2, GFAP and MBP , ultrastructure observing with electron microscope and engrafting to SCID mice for tumorigenesis test.The results were as following:Only a small subset of CD133+ glioma cells in glioma cell lines and fresh specimens from various pathologic grade could express stem cell markers CD133 and Nestin, view ultrastructure of a stem cell and be capacity of serial passage in culture. These CD133+ cells possese a marked capacity for multipotent differentiation and could differentiate into tumor cells expressing MAP2,β-TubulinⅢ, GFAP and MBP; When engrafted into SCID mice, they can generate and form tumors that phenotypically resembl the tumor from the patient. Therefore, the small fraction of CD133+ cells identified stem cells is BGSCs , so called as tumor stem cells in glioma.In Part 2 of the research, BGSCs in vitro were adopted as model. Genes and proteins expression of NOTCH-1 signaling pathway were detected during BGSCs proliferation and differentiation. Proteins expression of NOTCH-1 signaling pathway and cell type were also detected in glioma specimens and cell lines. The relationship between proliferation and differentiation of BGSCs has been analysed. On this basis, a retroviral-mediated expression system containing double strands DNA for RNA interference on human NOTCH-1 gene was constructed. Expression and function of NOTCH-1 signaling pathway in BGSCs were blocked by RNA interference. Immunity fluorescence technique, RT-PCR, Western blot and flow cytometry were used to evaluate effects on proliferation, differentiation, survival and tumorigenesis of BGSCs.The results were as following:1,Expression of NOTCH-1 and HES-1 protein were detected in glioma specimens and cell lines but small in normal adult brain tissue. More intensity of expression were detected in higher grade glioma.2,NOTCH-1 and HES-1 expressed in relativly differentiated and immature cells of CD133,MAP2, GFAP and MBP, especially in the cells of CD133. It implies that expression of NOTCH-1 and HES-1 may be correlated to iniatiation and maintenance of glioma differentiation.3,In proliferating BGSCs, NOTCH-1 mRNA and protein were detected strongly, which implies that the maintenance of strong NOTCH-1 signal may be correlated to stem cell state and survival.4,Expression of NOTCH-1 in differentiating BGSCs increased gradually, then tapered to disappearance; Expression of CD133 tapered too, but the expression of MAP2, GFAP and MBP appeared and increased gradually. Which suggests that proteins expression of may participate in initiation and phase control of BGSCs differentiation.In part 3 of the research, based on the part 2, A retroviral-mediated expression system containing double strands DNA for RNA interference on NOTCH-1 gene was successfully constructed. And by the technique of RNAi, we can found the change of the expression and function of NOTCH-1 signaling pathway. We can analyse the role of NOTCH-1 signaling pathway in BGSCs proliferation and differentiation.The results were as following:1,A retroviral-mediated expression system containing double strands DNA for RNA interference on NOTCH-1 gene was successfully constructed.2,When NOTCH-1 signaling was blocked by RNAi, proliferation of BGSCs were inhibited and viable cells decreased; Cells in G0/G1 phase decreased, in S phase was exhausted or held up, and apoptotic or necrotic cells increased. Which hints that NOTCH-1 signaling could promote proliferation, cell division and survival, and may implement by controlling cell cycle, promoting cell division and decreasing cell apoptosis or necrosis.3,When NOTCH-1 signaling was blocked by RNAi, differentiation of BGSCs were inhibited and dead cells incresed. At the group after RNAi, CD133,β-TubulinⅢ, GFAP and MBP positive cells ratio increased in RNAi group as compared with No-RNAi group and Negative-RNAi group. A inference could be drawn that NOTCH-1 signaling promoted differentiation and survival of BGSCs.It was general believed that the expression of genes and proteins of NOTCH-1 signaling pathway plays a key role in proliferating and differentiating BGSCs, Which promoted not only proliferation and survival but also differentation of BGSCs. It may implement by controlling cell cycle, promoting cell division and decreasing cell apoptosis or necrosis. However, NOTCH-1 signaling is not the only key signal pathways in proliferation, differentiation and survival of BGSCs, some other signals such as HH, WNT, EGF and bFGF may also be at work. It is supposed that numerous signals are involved in controlling the proliferation, differentiation and survival of BGSCs. Further deep and extensive studies on these key factors could show new light on control in that initiation, progression and recurrence of glioma at advanced level.