I.探討微核醣核酸-125b/LIN28B調控神經母細胞瘤惡性度之角色 II.建立微核醣核酸-125b基因剔除小鼠

Abstract

微核醣核酸（microRNA）為演化上具高度保留性之非轉譯型小核醣核酸分子，具有以轉錄後抑制方式控制大量標的基因表現之功能，涉及調控與分化、細胞週期控制、生長及細胞凋亡…等重要細胞活動。微核醣核酸在腫瘤細胞中的表現量，時常被發現異於正常細胞，且微核醣核酸在腫瘤中的角色可為抑癌或致癌基因。微核醣核酸-125b（miR-125b）是線蟲中負責調控蟲體發育時序關鍵基因lin-4的同源基因。微核醣核酸-125b在神經系統中高度表現，在誘發神經分化的過程中扮演重要的角色。目前微核醣核酸-125b在線蟲、果蠅及斑馬魚研究模型中已確定其與個體及神經系統發育息息相關，然而是否參與哺乳動物的發育機轉則尚不清楚。 神經母細胞瘤是兒童癌症中最常見的顱外惡性腫瘤。神經母細胞瘤起源於交感神經系統中發育分化異常的神經脊細胞。目前高危險群神經母細胞瘤患者經多方密集治療後，長期存活率仍僅達百分之四十。因此，了解神經母細胞瘤的致病機轉，都有助尋找新的治療標的及改善神經母細胞瘤患者的治療現況。在神經母細胞瘤的諸多致病機轉中，MYCN基因增幅是最為廣泛研究、且與臨床患者不良預後最具有相關性的指標分子之一。但其致病機轉以及如何影響神經母細胞瘤的惡性度，目前卻仍有許多未知之處。過去研究發現MYCN在細胞中，可廣泛性地抑制微核醣核酸的表現，包括微核醣核酸125b。此外，在預後較好的神經母細胞瘤患者中，其微核醣核酸-125b的表現量較高。微核醣核酸-125b的表現量來自兩個基因座─MIR125B1及MIR125B2。其中MIR125B1基因位於人類第十一號染色體長臂（11q24.1），在惡性度極高的神經母細胞瘤患者中，時常出現染色體缺失的區域。顯示微核醣核酸-125b可能在神經母細胞瘤扮演抑癌基因的角色。過去已知LIN28B是微核醣核酸125b的標的分子之一，在許多高惡性度的癌症高度表達，並參與細胞惡性癌變的過程。然而微核醣核酸-125b/LIN28B的調控機轉是否影響神經母細胞瘤的惡性度則仍不清楚。 本篇論文的第一部分，意在探討微核醣核酸-125b/LIN28B調控機轉對於神經母細胞瘤惡性度的影響，及LIN28B的表現量於神經母細胞瘤的臨床預後價值。第二部分我們希望透過基因剔除技術，建立微核醣核酸-125b基因剔除小鼠，以供未來深入研究微核醣核酸-125b對於生理調控功能，或人類疾病的動物模式。 在第一部份的研究中，我們發現微核醣核酸-125b在MYCN基因座增幅的細胞中表現量較低，而其下游分子LIN28B的表現量較高。利用慢病毒（Lentivirus）基因轉導的方式高度表現微核醣核酸-125b後，會抑制神經母細胞瘤的增生，並誘發其分化，且同時能抑制LIN28B的表現。我們分析神經母細胞瘤患者的腫瘤後，發現LIN28B在神經母細胞瘤組織的表達量，不僅可做為預測病患存活程度的獨立指標，且與MYCN基因增幅及MYCN表現量具有正相關性。以核糖核酸干擾技術抑制LIN28B的表現後，可誘發神經母細胞瘤進行分化、減緩細胞增生及抑制MYCN的表現量。當在細胞株中大量表現LIN28B後，可誘發MYCN的表現且增加癌細胞的非貼附性生長能力。顯示微核醣核酸125b/LIN28B/MYCN之訊息迴路對於神經母細胞瘤的惡性度，具有十分重要的角色。因此，未來若能利用針對此訊息路徑設計治療標的，可能有助於替神經母細胞瘤高危險群患者開發具有潛力的治療策略。 第二部份的研究中，我們成功產製Mir125b1及Mir125b2基因剔除小鼠。我們發現Mir125b1基因剔除小鼠出現出生後快速死亡的現象，且伴隨哺乳行為的異常表現，僅有極少數的Mir125b1基因剔除小鼠存活至三週，外觀也出現十分顯著的生長遲緩；而Mir125b2基因剔除小鼠卻沒有出現類似的表現型，我們也發現在控制食慾的神經中樞下視丘裡，70%的微核醣核酸-125b表現量來自Mir125b1基因座，顯示兩個基因座對於微核醣核酸-125b的貢獻度不盡相同。同時，我們也發現Mir125b1基因被剔除後，會誘發宿主基因2610203C20Rik表現量的上升。進一步探討發現2610203C20Rik的表現量提高是由於“in-cis”的影響，即使改以序列突變的方式破壞Mir125b1，仍無法抑止2610203C20Rik表現量上升的現象，顯示Mir125b1所形成的微核醣核酸環型結構，可能對於宿主基因扮演十分重要的調控角色。未來我們將進一步釐清Mir125b1基因剔除鼠的表現型及其可能的分子機轉，以發掘微核醣核酸-125b對於哺乳動物的生理功能。

MicroRNAs (miRNA) are phylogenetically conserved small non-coding RNAs known to post-transcriptionally regulate broad spectra of genes implicated in development, cell differentiation, cell cycle, cell growth and apoptosis. Aberrant expression of miRNA has emerged to be a common feature of cancer and miRNAs frequently serve as either tumor suppressor genes or oncogenes at different cancer types. MicroRNA-125b (miR-125b) is the mammalian ortholog of heterochronic gene from C. elegans, lin-4, which is a miRNA essential for normal temporal control of diverse development processes. MiR-125b is enriched in neural cells, and is up-regulated during all-trans retinoic acid (ATRA)-induced neuron differentiation. The role of miR-125b in development or central nervous system has been reported using several animal models such as C. elegans, D. melanogaster and D. rerio. However, whether miR-125b involved in the development of mammal remains elusive. Neuroblastoma is the most common extracranial tumor in pediatric malignancies. The tumor arises from the neural crest precursors of the sympathetic nervous system and represents aberrations of normal development programs. Long-term survival is achieved in only 40% of high-risk neuroblastoma despite intensive multimodal therapies. Therefore, identification of novel therapeutic targets and understanding the neuroblastoma pathogenesis are required to improve outcomes of neuroblastoma patients. MYCN amplification (MA) is a well-known oncogenic event in development of neuroblastoma, and is one of the strongest prognostic factors associated with unfavorable outcomes of patients. However, the mechanism of MYCN over-expression and how it promotes an aggressive disease remain largely unknown. MYCN suppresses the expression of a wide range of microRNAs, including miR-125b. Previous study indicated that higher expression of miR-125b was associated with low-risk neuroblastomas. MiR-125b was transcribed from two distinct genomic loci, MIR125B1 and MIR125B2. Interestingly, MIR125B1 gene located at 11q24.1, which was frequently deleted in high-risk neuroblastoma patients. Furthermore, LIN28B, a target of miR-125b, are frequently up-regulated in aggressive types of several cancers and facilitates cellular transfromations. These reports prompted us to hypothesize that miR-125b might serve as the tumor suppressor of neuroblastoma. However, the biological roles of microRNA-125b in neuroblastoma are undetermined. In the first part of this dissertation, we investigated miR-125b/LIN28B regulatory axis in modulating malignant phenotypes of neuroblastoma cells and evaluated the clinical significance of LIN28B in neuroblastoma. In the second part, we intended to establish miR-125b genetic knockout mice for further investigating the roles of miR-125b in physiological regulation or human diseases. At the first aim, we found the expression of miR-125b was lower in MYCN -amplified neuroblastoma cell lines, while the target gene, LIN28B, was relatively higher. Ectopic expression of miR-125b by lentiviral transduction in neuroblastoma cell line inhibited cell proliferation and induced neuronal differentiation accompanying with suppression of LIN28B. We evaluated clinical importance and prognostic value of LIN28B expression in primary neuroblastoma tumors and found that high LIN28B expression predicted a poor clinical outcome independent of other prognostic factors, including MYCN status, in neuroblastoma. Moreover, the expression of LIN28B was positively associated with MYCN amplification and mRNA expression level. Suppression of LIN28B by short hairpin RNAs (shRNAs) in MA neuroblastoma cell lines induced neuronal differentiation, reduced cell proliferation, and reduced MYCN expression. Inversely, ectopic LIN28B expression in MYCN non-amplified (MN) neuroblastoma cells induced MYCN expression and enhanced anchorage-independent growth. These results suggested that miR-125b/LIN28B/MYCN signaling circuit plays important role in modulating tumor malignancy. Thus, targeting the miR-125b/LIN28B/MYCN signaling axis may be a promising potential strategy for the treatment of aggressive neuroblastoma. At the second aim, we established Mir125b1 and Mir125b2 genetic knockout mice, respectively. We found that Mir125b1 knockout mice showed perinatal lethality accompanying with absence of feeding behavior whereas Mir125b2 knockout mice grew normally. Of the few Mir125b1 knockout mice survived for three-weeks, they presented severe growth retardation. In hypothalamus, which is the center of thirsty and hunger, the expression of miR-125b was mostly derived from Mir125b1 locus (70%).It suggested that Mir125b1 and Mir125b2 loci displayed different expression levels in given tissues. Furthermore, we observed an up-regulation of the host gene, 2610203C20Rik, in Mir125b1 knockout mice through “in-cis” regulation. Moreover, disruption of stem-loop structure of Mir125b1 through mutation knockin approach also upregulated 2610203C20Rik. It is suggested that maintenance of intact structure of Mir125b1 might be critical in regulating the expression of the host gene, 2610203C20Rik. In the future, we will further investigate the molecular mechanism of phenotypes of Mir125b1 knockout mice to elucidate the physiological roles of miR-125b in mammals.