探討神經膠質瘤C6 glioma cell自我更新及神經細胞分化

Studies of the self-renewal and the neural diferentiation of C6 glioma cells

趙娟娟(Chuan-Chuan Chao)

指導教授 ： 錢宗良

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.00678

摘要

惡性神經膠質瘤(malignant gliomas)是成人中最常見的原發性腦腫瘤。科學家嘗試著利用包括紫杉醇(taxol)在內的各種抗癌藥物治療膠質母細胞瘤，發現這些藥物不僅造成細胞死亡，同時也可以觀察到細胞的形態變化。然而治療效果卻不盡理想，或許是受到腫瘤幹細胞(cancer stem cells, CSC)的存在所致。
腫瘤幹細胞是腫瘤細胞中一群具有自我更新能力及並可以不斷形成腫瘤的細胞。將腫瘤幹細胞培養在含有bFGF及EGF的無血清培養基中，細胞則會形成具有神經幹細胞的球體(spheres)並具有表現神經幹細胞的標記—Nestin 蛋白。將細胞加以誘導分化則會形成表達bIII-tubulin, GFAP及CNPase等神經或是膠質標定蛋白的細胞。
MiRNA是真核生物中廣泛存在的一種RNA分子，其可藉由與其目標基因mRNA之3’-UTR互補結合，抑制轉錄後基因的表達，進而達到其基因調控的目的。除了在腫瘤幹細胞中扮演調控的角色外，這些miRNAs也與神經發育及腫瘤形成有關。
在本篇研究中，我們首先利用紫杉醇的刺激來觀察rat C6 glioma cells形態上的變化，同時並檢測經由紫杉醇刺激後glioma cells中其bIII-tubulin, GFAP及CNPase等神經標定蛋白的變化。結果發現C6 glioma cells受到紫杉醇的誘導引發神經分化。在神經膠質瘤的治療上，紫杉醇的給予提升了神經細胞的分化能力。此一“分化治療”或許可作為抗癌藥物在癌症治療上提供一潛在的治療方式。
其次，由於C6 glioma cells具有腫瘤幹細胞的三大特性—自我更新、多能分化及致癌能力，因此將C6 cells培養成球體細胞並進一步證明這些球體細胞具有腫瘤幹細胞的特性，結果發現C6 cells形成球體提升了多能分化的潛力並且受到IBMX的刺激後則會誘發神經分化。同時利用miRNA 微陣列進行球體細胞中miRNA的分析後發現miRNA-30c在球體細胞中高度表達。此外，在IBMX誘導神經分化的過程中，miRNA-30c藉由影響JAK-STAT3 訊息傳導路徑而抑制GFAP的表現。因此，miRNA-30c在腫瘤幹細胞其自我更新及神經分化的能力中扮演“調節者”的角色。

關鍵字

腫瘤幹細胞 ； 自我更新 ； 神經分化 ； 微小核醣核苷酸

並列摘要

Malignant gliomas are the most common intrinsic primary brain tumors in adults. Treating the cells from glioblastoma multiforme (GBM) with various anti-cancer drugs, not only cell death but also morphological changes have been observed. Scientists try to use anti-cancer drugs to treat tumors including taxol.
Cancer stem cells (CSCs) are a subpopulation of cells in the tumor that have self-renewal capacity and can give rise to heterogeneous cancer cells that constitute the tumor. CSCs were cultured in vitro as spheres using serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) and also showed remarkable similarities to normal neural stem cells (NSCs), expressing neural stem/progenitor markers such as Nestin, and, upon induction, could be differentiated to cells expressing neuronal or glial markers, including bIII-tubulin, glial fibrillary acidic protein (GFAP) and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase).
MicroRNAs (miRNAs) are noncoding sequences that act as “post-transcriptional” regulators that bind to complementary sequences in the 3′-untranslated regions of target mRNA transcripts by imperfect base-pairing, usually resulting in gene silencing. MiRNAs play a regulatory role in cancer stem-like cells including GBM. MiRNAs are also involved in neural development and tumor formation.
In this study, we first present the effects of taxol challenge on rat C6 glioma cells in the aspects of cellular morphological changes and differentiation. Characterizations of neural molecular markers including bIII-tubulin (for neurons), GFAP (for astrocytes) and CNPase (for oligodendrocytes) in the taxol-treated cells revealed that taxol might induce neural differentiation. In glioma cells, base on the fact that neural differentiation could be promoted by taxol administration, a potential therapeutic application of anti-cancer drugs on the differentiation of tumor cells was suggested. Secondary, it also has been proved that C6 cells have the attributes of self-renewal, multipotency, and tumorigenicity, which are the characteristics of cancer stem-like cells. In these studies, the ability of self-renewal was characterized by using “sphere formation” under culture conditions including bFGF, EGF, and B27 supplementation. These sphere cells were further confirmed as “cancer stem-like cells” based on their characteristics of multipotency and tumorigenicity.
In our studies, we demonstrated that a miRNA microarray served as a good platform for investigating which miRNA contributes in the processes of sphere formation and neural differentiation in this glioma cell model. Spheres were formed at first to enhance the potential for multipotency, and then neural differentiation was induced by 3-isobutyl-1-methylxanthine (IBMX) stimulation. Several miRNAs involved in sphere formation were identified by the miRNA microarray, and miRNA-30c was confirmed to play an important role in sphere formation. Furthermore, miRNA-30c suppressed the expression of GFAP by affecting the JAK-STAT3 pathway. These results suggest that miRNA-30c has a regulatory role in self-renewal and neural differentiation.

並列關鍵字

cancer stem cell ； self-renewal ； neural differentiation ； microRNA

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