微核糖核酸-146a和微核糖核酸-370以SOS1和GADD45b當作標的共同調控被腸病毒71型感染後的細胞凋亡

Abstract

腸病毒71型為一好發於亞洲地區的新興且致命之病原體。細胞凋亡為腸病毒71型感染後最主要的病理特徵．然而，對於腸病毒造成宿主細胞凋亡的分子機制仍未有透澈之研究報導．微核糖核酸(miRNAs)是一個近來發現的新分子，此分子可以透過後轉錄調控方式廣泛地影響生物機能，並且有報導指出微核糖核酸參與病毒致病機轉．本論文欲探討微核糖核酸與信息RNA(mRNA)是否協同調控腸病毒71型感染後所引發之宿主細胞凋亡．利用微核糖核酸與信息RNA表現圖譜加以生物資訊分析等策略，研究腸病毒71型感染後造成細胞凋亡之可能途徑．根據上述策略，可篩選出兩條與細胞凋亡相關之訊息傳遞路線，分別為BAD磷酸化(BAD phosphorylation)與p53所屬(p53-dependent)訊息傳遞．SOS1與GADD45β分別屬於BAD磷酸化與p53所屬訊息傳遞路線，且被預測為微核糖核酸-146a與微核糖核酸-370的標的．利用冷光報導基因試驗與西方墨點法證實微核糖核酸-146a與SOS1以及微核糖核酸-370與GADD45β之間存在負向調控關係．抑制微核糖核酸-146a可恢復SOS1表現量，並輕微減少腸病毒71型感染造成的細胞凋亡．相反地，異位表現微核糖核酸-370會降低因腸病毒71型感染上升的GADD45β的表現量，並減少細胞凋亡．最後，利用微核糖核酸-146a拮抗物與異位表現微核糖核酸-370可顯著減少腸病毒71型引起之細胞凋亡．在此，本研究證明腸病毒71型可藉由調控微核糖核酸表現造成宿主細胞凋亡的分子機制

Enterovirus 71 (EV71) is an emerging life-threatening pathogen particularly in the Asia-Pacific region. The major pathogenic feature in EV71-infected cells is apoptosis. However, which molecular mechanism mainly contributes to EV71-induced apoptosis is not investigated thoroughly. MiRNAs, the newly discovered molecules, govern a wide range of biological functions through post-transcriptional regulation and play roles in viral pathogenesis. Whether miRNAs and mRNAs coordinate to trigger host cell apoptosis in EV71 infection was investigated in this study. We conducted an apoptosis-oriented approach by using both mRNA and miRNA profiling and bioinformatic analysis. We identified two major apoptosis-associated signaling pathways, Bcl2 antagonist of cell death (BAD) phosphorylation and p53-dependent apoptosis pathways, in which Son of sevenless homolog 1 (SOS1) and Growth arrest and DNA damage-inducible protein 45β (GADD45β) were predicted as targets of miR-146a and miR-370, respectively. Luciferase reporter assays and Western blots demonstrated the negative regulation between miR-146a and SOS1 and between miR-370 and GADD45β. Silencing of miR-146a restored SOS1 expression and partially attenuated EV71 infection-induced apoptosis. Conversely, ectopic expression of miR-370 decreased virus infection-induced GADD45β expression and also diminished apoptosis. Finally, the co-expression of antagomiR-146a and miR-370 contributed to attenuating EV71 infection-induced apoptosis. Herein we clearly demonstrate a new mechanism that EV71-induced cell apoptosis is partly governed by altered miRNAs.