鈣網蛋白於神經母細胞瘤細胞株內調控血管內皮生長因子機制之研究

Abstract

神經母細胞瘤 (Neuroblastoma, NB) 為經常在幼兒及胎兒被發現的一種惡性腫瘤，具有存活率低和高轉移率 (Metastasis) 之特性。儘管有多個臨床相關的預測因子已經在神經母細胞瘤中被發現，其腫瘤形成的分子機制依然不清楚。鈣網蛋白 (Calreticulin, CRT) 為一種常儲存於內質網上的伴隨蛋白 (Chaperone protein)，它已經被證實在神經母細胞瘤的細胞分化以及腫瘤的形成的過程中扮演相當重要的角色。而在胃癌細胞中，它會經由促進血管內皮生長因子 (Vascular endothelial growth factor) 的表現來增強胃癌腫瘤內血管新生 (Angiogenesis) 之現象。在這篇研究當中，我們希望能夠了解鈣網蛋白和血管內皮生長因子之間的關聯性。我們先在神經母細胞瘤細胞株SK-N-DZ以及SH-SY5Y中利用表現質體和短髮夾核糖核酸去大量增進或抑制鈣網蛋白的表現，發現鈣網蛋白對於血管內皮生長因子有一正向調控之行為存在，且此現象在RNA層級和Protein層級皆有觀察到。此外，另一個已被證實在神經母細胞瘤中為血管內皮生長因子的上游的調控分子缺氧誘導因子-1α之表現量與鈣網蛋白也有一正向關之現象存在。藉由應用酵素結合免疫吸附分析的方式觀察到細胞株的培養基裡所含之血管內皮生長因子蛋白質的濃度確實會隨著鈣網蛋白過量表現或抑制而分別增加或減少。接著利用慢病毒的感染以及嘌呤霉素篩選出能經由四環黴素誘導鈣網蛋白表現之stNB-V1神經母細胞瘤細胞株。藉由異體移植的方式將此細胞株打入老鼠體內，再以餵食的方式讓小鼠攝入多西霉素來誘導鈣網蛋白之表現。結果觀察到鈣網蛋白被誘導表現之組別，其腫瘤體積會比控制組來的小上許多。此外誘導組別之腫瘤組織內的血管內皮生長因子、缺氧誘導因子-1α和分化標記分子GAP43及NSE2的表現量皆會提高，證實鈣網蛋白確實對於血管內皮生長因子以一正向調控之行為存在。

Neuroblastoma (NB) is a childhood cancer with a low survival rate and great potential for metastasis. Even though several clinically relevant prognostic markers have been identified, the molecular mechanism underlying the tumorigenesis of NB is still unknown. Calreticulin (CRT), an endoplasmic reticulum chaperone protein, is one of the biomarkers of NB. It has been suggested to play a critical role in the tumorigenesis and differentiation in NB. In gastric cancer cells, it was also found that CRT strongly enhances angiogenesis by promoting the expression of vascular endothelial growth factors (VEGFs). In this study, we aimed to investigate the correlation between the expression of CRT and of VEGF in NB. It was found that over-expression of CRT in SK-N-DZ and SH-SY5Y cells up-regulated VEGF expression at both the mRNA and protein levels. CRT RNAi efficiently suppresses CRT expression resulting in down-regulation of VEGF. By using ELISA analysis, CRT was shown to also significantly enhance VEGF-A expression in conditioned medium. The CRT-inducible stNB-V1 cell line has been established by the lentivirus transduction system and by puromycin selection. By using this inducible cell line, we will be able to clarify the underlying mechanism of how CRT regulates neuroblastoma metastasis. This CRT inducible NB cell line was applied to xenograft experiments. We found that xenograft tumors with induced CRT expression had much smaller sizes. VEGF-A had a much higher expression level in these tumor samples compared to control tumor samples. These results indicate that CRT plays a role in regulating VEGF-A expression, which may affect angiogenesis in NB. In vivo, a xenograft analysis will also be conducted to further confirm the role of CRT in neuroblastoma metastasis.