探討活化轉錄因子3調控microRNA 的分子機轉及臨床上的應用(1/3)

先前我們對微型核醣核酸(microRNA,or miRNA)在腎臟損傷的研究中，發現 miR-494 能調控 ATF3 基因的表現。然而，現今的報告也指出轉錄因子 (Transcriptional factor, TF)也能調控 microRNA 的表現，進而導致生理現象的改 變。轉錄因子和 microRNA之間相互的調控，顯示它們扮演重要的生理和病理角 色。然而有關 ATF3 是如何調控 microRNA，至今仍無相關的報告。我們先前的 數據利用腎臟上皮細胞經缺氧再灌流或大量表現 ATF3 後，收集細胞經由 microRNA array分析後發現，有許多的 microRNA增加或減少，其中 miR-16等的 表現量與 control 組有明顯的統計上差異(Fig.1)，我們更進一步利用 in vitro的實 驗大量表現 ATF3後發現 ATF3會促進 miR-16的表現(Fig.2)。從此初步的結果， 我們假設轉錄因子 ATF3 可能會調控 miR-16，進而影響腎臟在缺氧再灌流後的生 理病理現象。因此此三年期計畫主要的目的就是要釐清，在腎臟中，ATF3 到底 是如何調控 miR-16，且到底除了 miR-16 之外是否有其它的 miRs 能被 ATF3 調 控，進而影響腎臟的功能此外，在 ATF3調控下的 miR-16的 下游基因為何，進 而造成 ATF3在腎贓損傷中是扮演一保護的角色，也是我們研究的重點。下列為 未來三年要完成的目標: 1. 確認在 in vitro 情形下，ATF3與所能調控的 miR-16或其它 miRs 之間的關係 及分子機轉 (第一年)。 2. 確認在 in vivo 情形下，ATF3所調控的 miRs(如 miR-16)在腎臟生理或病理上 扮演一重要角色並確認這些 miRs 可作為 preclinical 的工具(第二年)。 我們之前的研究指出急性腎損傷病人的尿液中，miR-494, 16 及 ATF3 (exosome) (Fig.3)皆會增加。同時，這三者中 miR-16可作為偵測急性腎損傷的生 物標記，因此在第二年完成基礎研究後，我們要完成下列實驗以證明這些被 ATF3 所調控的 microRNA在臨床醫學上可以作為檢測腎損傷的 一個生物標記。 3. 確認 ATF3所調控的 miRs(如 miR-16)能在尿液中偵測出，並能作為檢測急性 腎損傷的生物標記 (第三年)

Our previous study showed that microRNA 494 (miR-494) can regulate the expression level of transcriptional factor, ATF3, in acute renal injury. In addition, recent reports also indicate that transcription factor can regulate microRNA (miR) expression resulting in the alteration of pathophysiological phenomena. The reciprocal modulation of transcription factors and miRs indicate that they play important pathophysiological roles. However, there are still no studies investigating the regulation of miRs by ATF3. Our previous data using microRNA array analysis revealed that many miRs, were up or down regulated when renal epithelium cells were treated under hypoxic/reoxygenation condition or overexpressed with ATF3 (Fig.1) indicating that the transcription factor, ATF3 can up or down-regulate miRs processing. We further observed that ATF3 overexpression resulted in the enhancement of miR-16 level in in vitro renal epithelium cells (Fig.2). These preliminary results suggest that not only miR-16 but other miRs may also be regulated by ATF3. We also propose that the altered expression of miRs may affect the pathophysiologic phenomenon in the kidneys after ischemia/reperfusion. In this three years proposal, we will evaluate which miRs in addition to miR-16 and down stream of miR-16 regulated genes may be regulated by ATF3, thereby affecting the function of the kidneys The goals to be completed for this 3-year proposal are as follows: 1. To characterize the miR-16 or the other miRs that will be regulated by ATF3 and to identify the molecular mechanisms involved in in vitro assay (First year). 2. To characterize the pathophysiological role of ATF3-regulated miRs (like miR-16) in the kidneys using in vivo assay, and to verify that the identified miRs can be used as a preclinical tool (Second year). Our previous studies demonstrated that mi-R494, 16 and ATF3 (exosome) (Fig.3) were all increased in the urine of patients with acute kidney injury. Furthermore, we also showed that miR-16 can be used as a urine biomarker of acute kidney injury patients (patented by Taipei Medical University). Therefore, based on the results from the first 2 years, we would like to learn further whether these ATF3-regulated miRs, besides miR-16, can also be used as biomarkers for monitoring patients suffering acute kidney injury. The goal of the 3rd year will be : 3. To confirm that ATF3-regulated miRs (like miR-16) can be used as biomarkers in patients with acute kidney injury (Third year). This study will provide not only a more detailed dissection of the relationship between transcriptional factor, ATF3, and miRs in kidney pathophysiology but may also provide these ATF3 regulated miRs as new therapeutic and diagnostic targets.