الفهرس 
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Abstract
The transcription factor REST is a transcription repressor that silences the terminal neuronal differentiation genes. Recently, REST has been shown to be associated with the core self renewal genes in mouse embryonic stem cells. In addition, REST was shown to play role in NSCs development. Meanwhile, GBM is highly aggressive brain tumor with poor prognosis, high incidence of tumor recurrence and resistance to chemo and radiotherapy. All of these properties are attributed to self renewing stem-like cells called GSCs. These cells share some characteristics with NSCs and responsible for initiation and maintenance of tumor. So these cells may be crucial cellular targets for curative brain tumor therapy. But this requires a thorough understanding of the mechanisms regulating the self renewal of these cells.
In this study, first, we examined if REST maintains the self renewal of normal mouse NSCs. Second, we studied if REST regulates patient derived GSCs self renewal capacity and tumor forming ability. Moreover, we tried to get more insight into the mechanisms regulating the role played by REST.
In order to study role of REST in NSCs, we isolated the primary E12 and E14 NSCs from brains of mice embryos. We used WT and REST-eGFP mice. These are transgenic mice where GFP is expressed under REST promoter. First, we showed that primary neurosphere isolated from E12 and E14 cortices of these transgenic mice coexpress REST, GFP and NSC marker Sox2 or Nestin under proliferation conditions. Upon differentiation, the expression of REST, GFP together with Sox2 or Nestin went down with concomitant expression of the three neural lineage differentiation markers β-tub (neuronal), MBP, (oligodendrocytic) and GFAP (astrocytic) lineages i.e. these cells are multipotent. This indicates that REST is associated with stem cells in vitro.
Second we sorted the GFP+ve cells (REST+ve cells) using FACS analysis from both E12 and E14 NSCs and studied the characteristics of these cells. We found that REST expressing NSCs fulfill NSCs characteristics; they can self-renew for several passages and undergo symmetric and asymmetric divisions besides their multipotency.
In order to examine if REST is important for maintaining the self renewal capacity of NSCs, we knocked down REST in these NSCs by either shRNA for E12 or siRNA for E14 NSCs. Consequently, these cells exhibited decreased self renewal capacity indicated by neurospheres assay and loss of self renewal markers e.g. Sox2 or Bmi-1 expression. To gain more insight into the mechanisms by which REST mediated its role, we further examined 2 REST known targets miRNAs; miR21 and miR124 in NSCs upon REST knockdown. Interestingly, both miRNA were elevated in E12NSCs upon REST knockdown. However, only miR124, but not miR21, was significantly elevated in E14NSCs.
Thus, our results illustrated a new direct role of REST in maintaining stemness of NSCs. In addition, REST might be mediating its role through a miRNA dependent regulation of self renewal molecules like Sox-2 or Bmi-1.
Then, we tried to expand our findings on NSCs to GSCs based on the similarities between both of them. We examined the REST expression on different primary GSCs cell lines derived from GBM patient’s tumors. We found that GSCs derived from GBM patient samples can be divided into high REST (HR-GSCs) and low REST (LR-GSCs) subtypes according to REST protein expression. Functional assays and protein expression assays revealed that HR-GSCs have more self renewal capacity and express a mixture of NSC self renewal and differentiation markers. Although both groups produced human GBM-like tumors when injected into nude mice, the HR-GSCs produced more infiltrative tumors compared to circumscribed tumors characteristic of LR-GSCs.
To further demonstrate the role of REST in these GSCs, stable knock down of REST using shRNA in HR-GSCs not only decreased the self renewal of these cells but also it produced more survival in transplanted mice. Conversely, forced expression of REST in LR-GSCs increased self renewal of these cells, decreased survival of the transplanted mice and produced brain tumors with increased infiltration and histopathologically similar to the HR-GSCs tumors.
Thus we propose that REST is associated with maintaining the NSCs self renewal. The mechanism behind this may be through microRNA based regulation. In addition we found that REST regulates certain subtype of GBM by maintaining self renewal of GSCs. Our results suggest that REST, through maintaining the self renewal and tumorigenicity of stem like cells in GBM, can be a potential therapeutic target for GSCs in GBM tumors.