鈣網蛋白調控血管內皮生長因子之mRNA穩定性

Abstract

鈣網蛋白 (Calreticulin, CRT)是一個主要位於內質網的多功能伴護蛋白 (Chaperone protein)。之前有研究指出，在不同的癌症組織切片中，皆發現CRT會大量表現；另外也發現在膀胱癌J82細胞株中，CRT會透過穩定FUT1的mRNA，進而促進腫瘤的生成以及轉移。此外，大量表現CRT亦會增加在不同的癌症模式中被廣泛認為是血管新生主要調控者的血管內皮生長因子A的表現。在本研究中，我們嘗試在膀胱癌J82細胞株、胃癌AGS細胞株和前列腺癌PC3細胞株之癌症模式中，釐清CRT是否調控VEGF-A的mRNA 穩定性。我們發現，當減少CRT的表現後，VEGF-A的mRNA半衰期會明顯地縮短，進而抑制VEGF-A的mRNA以及蛋白質的表現。我們進一步的利用雙冷光試驗(dual-luciferase assay)證實CRT是透過辨認位於VEGF-A的3端為轉譯區 (3’ untranslated region, 3’UTR) 上之ARE片段(AU-rich element) 去調控VEGF-A的mRNA穩定性。確實，在生物素沉澱實驗 (biotin pull-down assay) 和液相層析串聯式質譜儀 (LC-MS/MS) 的結果中，證實CRT會與VEGF-A的ARE片段結合形成RNA-蛋白質複合體。綜合以上結果，本研究提供了一個全新的假說，在不同的癌症模式中，CRT會透過與位於VEGF-A的3端為轉譯區上之ARE片段結合而穩定VEGF-A的mRNA，而進一步促進癌症的進程以及有利於轉移之內皮細胞的血管新生。

Calreticulin (CRT) is a multifunctional chaperone protein, which mainly locates on the membrane of endoplasmic reticulum (ER). Previous studies suggested that expression of CRT is up-regulated in various types of cancers and promotes tumorigenesis and metastasis through stabilizing fucosyltransferase 1 (FUT-1) mRNA in bladder cancer cell line J82. Besides, overexpressed CRT up-regulates the expression of Vascular Endothelial Growth Factor A (VEGF-A), which is widely considered as a major regulator for angiogenesis in different cancer models. In this study, we therefore attempt to clarify whether CRT regulates VEGF-A mRNA stability. We observed that depletion of CRT results in a significant decrease of mRNA stability of VEGF-A following by suppression of both VEGF-A RNA and protein levels in bladder cancer, gastric cancer, and prostate cancer. Further, dual luciferase assay was performed to demonstrate that CRT recognized AU-Rich Element (ARE) of VEGF-A in the 3’UTR to regulate stability of VEGF-A mRNA. Indeed, the results of biotin pull-down assay showed that CRT combines to ARE region of VEGF-A to form RNA-protein complex which was confirmed by additional liquid chromatography–mass spectrometry (LC-MS/MS). In conclusion, this study provides a novel machanism that CRT forms mRNA-protein complex with ARE at the 3’UTR of VEGF-A to increase VEGF-A mRNA stability in distinct cancer cell lines, which CRT enhances cancer progression and endothelial cells angiogenesis for metastasis.