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標題: | 在SH-SY5Y細胞中利用CRISPR/CAS9技術探討特定微核醣核酸在神經母細胞瘤細胞中的作用 Investigating the effects of specific microRNAs in neuroblastoma using CRISPR/CAS9-mediated gene targeting in SH-SY5Y cells |
作者: | Ke-Wei Wang 王克巍 |
指導教授: | 胡忠怡 |
關鍵字: | 神經母細胞瘤,微核醣核酸,抑癌因子,CRISPR/Cas9基因編輯技術, neuroblastoma,microRNA,tumor suppressor,CRISPR/Cas9 system, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 神經母細胞瘤 (Neuroblastoma, NB)是常見的兒童顱外惡性腫瘤,約造成兒童癌症死亡的12%;根據其組織特徵、轉錄體和分化的特性,被認為是由於腎上腺交感神經前驅細胞異常分化增生所導致。NB為異質性很高的腫瘤,約20~25% 的NB病人腫瘤具有MYCN基因增幅,而MYCN是否發生基因增幅為目前所知NB最重要的預後不良因子。從大規模基因分析數據中可得知高度惡性NB基因外顯子突變率低,且較少有反覆出現的突變基因,表示在基因突變外的異常基因調控網絡對於NB發展以及進程上扮演重要角色。目前對於高度惡性的NB病患是以高劑量的化療/放療/手術切除等多管道方式進行治療,但在半數病患治療成效仍不甚理想。
微核醣核酸mircoRNA為長約20~23個核苷酸短片段、內生性的單股核糖核酸,主要功能為透過轉譯後調控抑制標的基因的表現。過去研究發現microRNA的表現與多種癌症的形成相關,且在不同系統癌症中可能扮演抑癌因子或致癌因子不同角色。我們先前系列研究中發現:在NB病患中腫瘤的miR-125b, miR-30d低表現量與較差的臨床預後相關。且在MYCN amplified NB細胞株SKNDZ, SKNBE中miR-125b, miR-30d 的表現量低於MYCN non-amplified 細胞株SKNSH, SH-SY5Y。過去在SKNDZ、SKNBE細胞株中利用慢病毒轉導過表現miR-125b, miR-30d後觀察到細胞增生及非貼附生長受抑制、遷移及侵襲能力降低,並且細胞內的ATP產生增高,暗示了miR30d和miR125b在MYCN-A NB扮演抑癌因子的角色並可能影響細胞內能量代謝。可惜因技術困難尚未能成功有效抑制miRNA的表現以確證miR-125b與miR-30d表現受抑制下是否影響NB腫瘤細胞的惡性表現型。 CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)是一種新興的基因剪輯工具,可以高特異性的在活細胞中編輯我們有興趣的基因; 在本研究中我們成功利用雙gRNA引導的CRISPR/Cas9技術在SH-SY5Y NB細胞株中對MIR125B1或MIR30D基因座進行編修,且成功篩選出標的細胞,得以有效抑制miR-125b或miR-30d表現。在二種經編修的標的細胞中均發現細胞生長速度上升、細胞非貼附生長能力上升、遷移能力上升以及ATP生成量減少等現象,確證miR-125b, miR-30d在MYCN-NA NB腫瘤中亦扮演tumor suppressor的角色並可能涉及細胞中的能量代謝。本研究並針對gRNA可能引發的脫靶效應進行探查,結果並未發現脫靶效應發生。MIR125B1及MIR30D標的細胞的產製將可運用於後續探索miR-125b, miR-30d在NB腫瘤生成中的調控網絡以及下游影響的基因,並找尋惡性NB可能的治療標的。 Neuroblastoma (NB) is a common extracranial malignant tumor in children, accounting for about 12% of pediatric cancer mortality. It is characterized by abnormal differentiation and proliferation of adrenal sympathetic nerve precursor cells. NB is a highly heterogeneous in clinical settings. About 20-25% of NB patients have MYCN-amplification in the tumor, which is currently known as the most important prognostic factor of NB’s disease progression. A large-scaled genetic study on high risk NBs revealed a low median exonic mutation and notably few recurrent mutated genes, indicating aberrant gene regulation network beyond genetic mutation plays an important role in tumorgenesis and tumor progression in NB. Parients with high-risk NB are currently treated with high-dose chemotherapies/ radiotherapy and surgical resection, however, the treatment outcome remained poor in half of these patients. MircoRNAs are endogenous small RNAs of 20~23 nucleotides that can transcriptionally repress their target genes. Previous reports showed that microRNA can act as a tumor suppressor or be oncogenic in different context. We previously showed lower miR-125b or miR-30d expression in primary NB tumors correlated to adverse clinical outcome. Among NB cell lines, miR125b and miR-30d expression are lower in MYCN-amplified (MYCN-A) SKNDZ and SKNBE than in MYCN-non amplified (MYCN-NA) SKNSH and SH-SY5Y. Enforced overexpression of miR-125b or miR-30d in SKNDZ and SKNBE cells inhibited cellular proliferation, reduced anchorage-independent cell growth, migration and invasion ability while increased glucose uptake and ATP production. These data indicate that miR125b and miR-30d play as tumor suppressors in NB tumors and may be involved in the regulation of energy metabolism. However, we have not yet confirmed the tumor suppressive effects of these two microRNAs in the experimental settings with reduced miR-125b, miR-30d expression in NB cells, due to problems encountered in anti-miR technique. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/ CRISPR-associated proteins) system is a newly emerged gene editing tool used for specific gene alteration in living cells. We successfully targeted MIR125B1, MIR30D in MYCN-NA SH-SY5Y cell line using dual gRNA-guided CRISPR/ Cas9 system and obtained gene-targeted cell clones with marked miR-125b or miR-30d repression. These clones showed enhanced cellular proliferation, anchorage-independent cell growth, migration ability and decreased ATP production, as compared to the control cells, which verify the hypothesis that miR-125b and miR-30d play as tumor suppressors in NB tumors and is involved in the regulation of energy metabolism. We also used RGEN software to predict off-target sites of each gRNA and PCR/sequecing to rule out the possibility of off-target damage of the tested cells. In the future, we will continue to explore the gene regulatory network of miR-125b and miR-30d in tumorigenesis and its downstream regulatory genes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78636 |
DOI: | 10.6342/NTU201903306 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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