鈣網蛋白在前列腺癌細胞中藉由3端未轉錄區上AU-Rich序列調控mRNA穩定性之研究

Abstract

調控訊息RNA (Messenger RNA, mRNA) 的穩定性為一種重要的轉錄後修飾，此調控決定 mRNA 之命運且影響許多細胞功能。先前的研究指出，mRNA的穩定性受到位於 mRNA 3端為轉錄區 (3’UTR) 上的 ARE (AU-Rich Element) 片段與其結合蛋白 (ARE-Binding Protein, ARE-BP) 的結合所影響。鈣網蛋白 (Calreticulin, CRT) 為一多功能伴護蛋白。過去的研究中指出，CRT 具有與 3’UTR 結合的能力。此外，CRT的表現量與血管內皮生長因子A (VEGF-A) 和 β1 型整合蛋白 (β1 integrin) 的表現量呈現正相關。然而，CRT 與 VEGF-A 以及β1 integrin 之間的調控機制尚不明確。因此本篇研究目的為探討 CRT 是否經由與 ARE 片段的結合調控mRNA穩定性，進而影響蛋白質的表現量。實驗的結果顯示，在前列腺癌細胞中抑制 CRT 的表現量，明顯地降低VEGF-A 以及β1 integrin的mRNA穩定性，進而降低 VEGF-A 以及β1 integrin 之表現量。同時，序列比對結果顯示在VEGF-A和β1 integrin的3’UTR上皆含有ARE片段。報導基因冷光活性試驗 (Luciferase Reporter Assay) 的結果顯示，CRT確實經由ARE片段來調控mRNA的穩定性。 RNA免疫沉澱法 (RNA-Immunoprecipitation)以及RNA電泳膠遲緩分析 (Electrophoretic Mobility Shift assay, EMSA) 的結果顯示，CRT 不直接與mRNA做結合。總而言之，CRT透過與3’UTR上的ARE片段，穩定 VEGF-A以及β1 integrin mRNA，進而調控蛋白質的表現量。

Messenger RNA (mRNA) stability is a post-transcriptional regulation that determines mRNA fate and influences multiple cellular functions. Previous studies have demonstrated that AU-rich element (ARE) in 3’-untranslated region (3’-UTR) of mRNA is critical for mRNA stability via interaction with ARE-binding proteins (ARE-BPs). Chaperone protein calreticulin (CRT) has been reported to bind the 3’-UTR. The expression level of CRT is positively correlated with the expressions of angiogenic factor vascular endothelial growth factor-A (VEGF-A) and β1 integrin. However, the detailed regulatory mechanism of CRT on VEGF-A and β1 integrin expressions remains elusive. Therefore, this study aimed to investigate whether CRT regulates mRNA stability through interaction with ARE. Our results showed that depletion of CRT significantly destabilized VEGF-A and β1 integrin mRNA in PC-3 human prostate cancer cells, and subsequently decreased their expression in both mRNA and protein levels. Sequence analysis showed that ARE in found in the 3’UTR of both mRNA. The result of Luciferase reporter assay demonstrated that CRT interacts with ARE. RNA-immunoprecipitation analysis and electrophoretic mobility shift assay (EMSA) demonstrated that CRT indirectly interacts with mRNA. In summary, this study showed that CRT regulates mRNA stability through interacting with ARE at the 3’-UTR of mRNA.