微核醣核酸miR-1在胃腸基質瘤進程的角色

Abstract

前言:胃腸基質瘤(Gastrointestinal Stromal Tumors, GISTs ) 是因為c-kit基因突變造成c-kit功能異常活化的胃腸道間葉腫瘤。胃腸基質瘤的侵犯能力可分級成從良性(低風險性)變化到惡性(高風險性)，但是控制胃腸基質瘤進展的機制尚不清楚。本篇研究目的為，探究微核醣核酸-1 (MicroRNA-1, miR-1) 是否透過標的蛋白B細胞淋巴瘤-2(B Cell Lymphoma-2, Bcl-2)而影響胃腸基質瘤的進展。 材料與方法:收集從臨床手術切除的人類胃腸基質瘤檢體，並依腫瘤的大小和細胞有絲分裂數目分成低風險 (LR)和高風險 (HR) 兩組。將檢體進行微陣列分析(Microarray analysis)和即時定量聚合酶連鎖反應(Quantitative real-time ,Q-PCR)以比較兩組之間微核醣核酸的表現差異，並以西方點墨法(Western blot analysis) 和免疫化學染色法(immunohistochemistry staining, IHC staining) 分析兩組間Bcl-2的表現，再以TUNEL assay 比較兩組間細胞凋亡的情形。為了瞭解miR-1對Bcl-2的調控角色，以微核醣核酸-1抑制物處理人類胚胎腎臟細胞株(human embryonic kidney cell line, HEK293)並觀察細胞株Bcl-2表現量的變化。 結果:微陣列分析和即時定量聚合酶連鎖反應顯示，在低風險胃腸基質瘤的臨床檢體中，微核醣核酸-1的表現量高於高風險胃腸基質瘤十倍。相反地，西方點墨法和免疫化學染色法顯示，B細胞淋巴瘤-2的表現量在高風險胃腸基質瘤較高。藉由轉染微核醣核酸-1抑制物進入人類胚胎腎臟細胞株，使微核醣核酸-1失去功能後，會增加細胞株中的Bcl-2表現量(控制組：0.0808±0.06 v.s實驗組：0.2418±0.05，p<0.05)。 結論:吾人研究發現高風險胃腸基質瘤和低風險胃腸基質瘤相比，表現較少的微核醣核酸-1和較多的B細胞淋巴瘤-2。降低細胞中微核醣核酸-1的表現，則會增加B細胞淋巴瘤-2的蛋白質表現量。這些結果顯示微核醣核酸-1可能藉由標的B細胞淋巴瘤-2調控胃腸基質瘤的細胞凋亡與腫瘤進展。

Introduction: Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors with gain-of-function mutation of c-kit genes in the gastrointestinal (GI) tract. The aggressiveness of GIST varies from benign (low-risk) to malignant (high risk) spectrum. The mechanisms controlling the progression of GISTs have not been explored. The aim of this study is to investigate that whether microRNA-1 (miR-1) play a role in the progression of GISTs by targeting the anti-apoptosis pathway. Methods: Clinical specimens from resected GISTs tumors were collected and grouped into low-risk (LR) and high-risk (HR) according to the tumor size and mitotic ratio. Microarray analysis, Quantitative real-time PCR (Q-PCR), Western blot analysis, immunohistochemistry staining (IHC staining), and TUNEL assay were performed in these two groups of tumors. To further determine the correlation with Bcl-2 and miR-1 in vitro, we treated HEK293 (human embryonic kidney cell line) with miR-1 inhibitor to observe the difference of Bcl-2 expression. Results: Microarray profiling and Q-PCR validation showed that miR-1 expression is tenfold higher in clinical specimen of LR GISTs than those of HR GISTs. On the contrary, Western blot analysis and immunohistochemistry staining showed that Bcl-2 expression is higher in HR group. In vitro, loss-of-miR-1 function by transfecting a miR-1 inhibitor into HEK293 cell increases Bcl-2 expression. Conclusions: High risk GISTs express less miR-1 and more Bcl-2 than low-risk GISTs do. In vitro, knockdown of miR-1 effectively enhances bcl-2 protein levels. Suggest that miR-1 regulates the progression and cell apoptosis of GISTs by targeting the Bcl-2.