N6-甲基腺苷修飾促進鈣網蛋白穩定血管內皮生長因子A mRNA之功能

Abstract

血管新生主要由血管內皮生長因子-A（VEGF-A）及其受器所調控，並在癌症的進程以及惡化扮演著極其重要的角色。在胃癌以及其他癌症中，越高的VEGF-A表現量將導致越差的預後狀況。因此，了解VEGF-A的詳細調控機制，將可提供癌症在臨床上新的治療策略。N6-甲基腺苷（N6-methyladenosine, m6A）甲基化為一種在訊息RNA（Messenger RNA, mRNA）上最常見的修飾之一。此甲基化修飾會調控RNA結合蛋白與RNA的結合，進而影響mRNA的表現量。在實驗室先前的研究中證實，鈣網蛋白（Calreticulin, CRT）會間接地結合到VEGF-A的3’未轉譯區（3'UTR）上調控其mRNA穩定性。因此本研究欲探討m6A甲基化在CRT調控VEGF-A mRNA穩定性的機制中所扮演的角色。我們首先透過MeRIP定量檢測驗證在胃癌細胞株AGS中其VEGF-A mRNA上帶有m6A甲基化修飾。藉由小分子干擾RNA （short interfering RNA, siRNA）抑制Mettl3與Mettl14所組成的N6-腺苷-甲基轉移酶複合體後，VEGF-A mRNA上的m6A甲基化修飾減少，造成mRNA的穩定性下降，進而影響mRNA以及蛋白質的表現。在報導基因冷光活性試驗（luciferase reporter assay）結果中，我們發現m6A甲基化修飾確實參與到CRT調控VEGF-A mRNA的機制中。RNA免疫沉澱（RNA-immunoprecipitation, RNA-IP）的結果顯示，當VEGF-A mRNA上的m6A甲基化修飾減少後，CRT與VEGF-A mRNA的結合顯著下降。在本研究中，我們釐清m6A甲基化修飾透過影響CRT與VEGF-A mRNA的結合，進而影響其調控VEGF-A mRNA的穩定性。

Vascular endothelial growth factor-A (VEGF-A) plays a critical role in the angiogenesis and progression of cancers. It is shown that AU rich elements (ARE) in 3’UTR of VEGF-A mRNA governs VEGF-A mRNA stability in manner of post-transcriptional regulation. Furthermore, our previous studies demonstrated that Calreticulin (CRT) acts as RNA binding protein (RBP) to stabilize VEGF-A mRNA via indirectly binding to ARE in 3’ untranslated regions (3’UTRs) of VEGF-A mRNA. CRT is a multifunctional chaperone protein, and elevated levels of VEGF-A and CRT are both highly correlated with poor prognosis in gastric cancers. Notably, RNA N6-methyladenosine (m6A) methylation is reported to promote the progression of gastric cancers. m6A modification has been reported to stabilize mRNA via mediating the interaction of mRNA with RBP. Therefore, this study aimed to investigate if m6A methylation regulates binding of CRT to VEGF-A mRNA. Our MeRIP results evidenced that VEGF-A mRNA contained m6A methylation sites in gastric cancer. The depletion of Mettl3 and Mettl14, m6A methyltransferase complex, destabilized VEGF-A mRNA in AGS, and thus decreased its RNA and protein expression. Luciferase reporter assay suggested that m6A methylation regulates VEGF-A mRNA via the CRT-dependent pathway. Moreover, RNA-immunoprecipitation (RNA-IP) analysis showed that depletion of m6A methylation significantly decreased the binding affinity of CRT to VEGF-A mRNA, and thus interfered the CRT regulation on VEGF-A mRNA stability. In summary, these findings identify the m6A methylation in 3’UTR as a key factor for CRT to recognize VEGF-A mRNA, and regulate VEGF-A mRNA stability.