Calreticulin在Notch調控之神經母細胞瘤分化所扮演的角色

Abstract

神經母細胞瘤為新生兒神經脊細胞不正常分化的惡性腫瘤。然而神經母細胞瘤的致癌機制非常複雜，有些病例不需要經過任何治療神經母細胞瘤就會自行分化而痊癒。在神經母細胞瘤的癌化過程中，Notch是很重要的關鍵蛋白；而神經母細胞瘤良性預後因子之一的Calreticulin （CRT），在近期的研究中被發現可能與神經母細胞瘤的分化有很密切的關連。根據我們實驗室之前的研究，抑制神經母細胞瘤中的Notch會促進神經母細胞瘤的分化，且細胞中CRT蛋白質的表現量會上升。但是，在神經母細胞瘤中Notch分子路徑與CRT的關係仍然沒有被仔細研究過。我們進一步假設CRT的mRNA表現也會受到Notch分子路徑的影響。我們首先利用Real-time PCR測量神經母細胞瘤細胞株SH-SY5Y在抑制Notch後，CRT mRNA在細胞內的表現量。如同預期，在抑制Notch的SH- SY5Y中CRT的mRNA表現會有顯著地增加。同時更進一步發現，位於CRT ORF 5’上游啟動子序列從-500到-2000之間可能包含有Notch signaling-dependent的調控序列。為了進一步證明CRT在神經母細胞瘤分化中是否扮演一個重要的角色，我們將SH-SY5Y中的CRT以RNAi的方法減低其表現量的情況下，再以Notch分子傳遞訊息抑制劑或視黃酸(retinoic acid)引導神經母細胞瘤的分化，結果發現在CRT表現減低的細胞，其被誘導分化的程度都有顯著減少的現象，可見CRT在神經母細胞瘤分化路徑中可能扮演一個很重要的樞紐。因此以我們研究的結果可以推論，Notch分子路徑會經由調控CRT的轉錄來影響神經母細胞瘤的分化。

Neuroblastoma was a solid tumor originated from undifferentiated neural crest cells. However, some neuroblastoma can regress without any remedy, indicating the pathological complexity of this tumor. Notch is a surface receptor that plays a key role in the tumorgenesis of neuroblastoma. We have previousely demonstrated that inhibition of Notch signaling causes neuronal differentiation of neuroblastoma, and induces the expression of a favorable prognostic marker, calreticulin (CRT). We thus proposed to address the molecular mechanism underlying the Notch-dependent regulation of CRT expression in neuroblastoma cells. To determine whether transcription of CRT is subject to the regulation by Notch signaling, by using real-time PCR, we demonstrated that the CRT mRNA levels were significantly enhanced by the inhibition of Notch in a neuroblastoma cell line SH-SY5Y. Moreover, we identified a putative binding motif of HERP, a Notch responsive gene, within the CRT promoter region. Consistent with this finding, a luciferase reporter gene construct encoding this CRT promoter sequence was shown to be responsive to the inhibition of Notch signaling. To further test whether CRT indeed plays an important role in the differentiation of neuroblastoma cells, we employed a RNAi approach to down-regulate CRT expression in SH-SY5Y cells and found that the down-regulation of CRT significantly hindered the neuronal differentiation of neuroblastoma cells induced by a Notch signal inhibitor and retinoic acid. Our findings strongly suggest that CRT critically involves in a mechanism governing the Notch signaling mediated differentiation of neuroblastoma through transcriptional regulation of CRT gene expression.