請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70408
標題: | 探討長鏈非編碼核糖核酸SNHG1在人類神經母細胞瘤的癌化機制 Long Non-coding RNA SNHG1 in the Regulation of Neuroblastoma Tumorigenesis |
作者: | Shih-Han Lin 林詩涵 |
指導教授: | 阮雪芬(Hsueh-Fen Juan) |
關鍵字: | 神經母細胞瘤,MYCN,長鏈非編碼核醣核酸,SNHG1,液相層析串聯式質譜儀,核醣核酸測序,細胞遷移, Neuroblastoma,MYCN,Long non-coding RNA,SNHG1,LC-MS/MS,RNA-seq,cell migration, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 神經母細胞瘤(neuroblastoma)是一種好發於兒童的癌症,為一種常見的胚胎腫瘤,源自於未分化的神經嵴細胞。大約25%的神經母細胞瘤患者發生MYCN轉錄基因異常放大,導致高風險疾病和不良預後。近年來,許多研究指出長鏈非編碼核醣核酸(long noncoding RNA)在腫瘤的形成過程中會扮演許多重要的角色。長鏈非編碼核醣核酸為長200餘鹼基對似訊息核醣核酸,但不會轉譯出蛋白質,其功能之一為引導蛋白質(例如:轉錄因子)到它的標的蛋白質。然而,由MYCN轉錄基因異常放大所調控的長鏈非編碼核醣核酸,其與神經母細胞瘤的高風險族群及不良預後之間的關係仍不明確。在我們實驗室之前的研究中,從MYCN轉錄基因擴增(n=92)及MYCN轉錄基因非擴增(n=401)的腫瘤中,結合微陣列(microarray)與核醣核酸測序(RNA-sequencing) 的長鏈非編碼核醣核酸基因表現調控資料庫,進行網絡整合分析,我們發現small nucleolar RNA host gene 1 (SNHG1)的表現量差異和MYCN轉錄基因異常放大表現量呈現高度正相關性,並利用即時聚合酶鍊式反應驗證之。在本研究中,為了研究SNHG1的調控機制,我們運用小分子干擾核糖核酸 (siRNA) 以及基因編輯技術CRISPR/Cas9在神經母細胞瘤細胞中敲落或刪除SNHG1的基因位點,減弱或剔除SNHG1會影響細胞增生。我們發現MYCN會調控SNHG1的啟動子,且抑制MYCN的表現量後,SNHG1的表現量也隨之下降;另外,有先前研究指出,SNHG1會調控MYC,其和MYCN有補償作用,也就是說,敲落SNHG1的表現量後,其會抑制MYC的表現量,但卻促進MYCN的表現量,這也是敲落SNHG1的表現量後,造成細胞大量增生的原因之一。我們進一步整合SNHG1干預的蛋白體和轉錄體學去了解其下游調控因子及其參與的生物途徑,研究發現SNHG1影響的功能集中在細胞增殖,細胞粘附和細胞遷移。我們也以細胞遷移分析實驗、即時聚合酶鍊式反應和西方墨點法證實剔除SNHG1會增加與遷移有關的蛋白質L1CAM, EGFR 和PRKCA表現,而促進細胞遷移能力。這些結果表示SNHG1參與調節MYCN和MYC的反饋迴路中,並促進有助於神經母細胞瘤腫瘤發生的細胞遷移。 Neuroblastoma is a common embryonal tumor originating from undifferentiated neural crest cells. Approximately 25% patients with neuroblastoma have MYCN oncogene amplification resulting in high-risk disease and poor prognosis. Recently, long noncoding RNAs (lncRNAs) are reported to have various roles in tumorigenesis. Long noncoding RNAs constitute major proportion of the cellular transcripts with no coding capacity. One of their functions is to guide transcription factors to the target genes and facilitate gene expression. However, lncRNAs that are altered by MYCN amplification, associated with high-risk, and disease prognosis remain unclear. We performed a network-based integrative analysis from both microarray and RNA-seq lncRNA expression profiles between MYCN amplified (n = 92) and MYCN non-amplified (n = 401) tumor subtypes. We identified the differentially expressed lncRNA small nucleolar RNA host gene 1 (SNHG1) to be significantly correlated with MYCN-amplification, and was further validated by RT-qPCR. Here, to study the regulatory function by SNHG1, we performed small interfering RNAs (siRNA) targeting SNHG1 and CRISPR/Cas9 editing to delete the genomic locus of SNHG1 in neuroblastoma cells, while knockdown or knockout of SNHG1 increased cell proliferation. We found MYCN binds to the promoter of SNHG1 and silencing MYCN downregulated SNHG1 expression. Additionally, previous studies have reported that SNHG1 regulated MYC which compensates MYCN. Thence, knockdown of SNHG1 would decrease MYC expression but enhance MYCN expression, which was the reason why knockdown of SNHG1 increased cell proliferation. We further integrated SNHG1-mediated proteomics and transcriptomics to reveal the downstream regulators and its biological processes. Our investigation found the functional enrichment on cell proliferation, cell adhesion and cell migration. Moreover, we confirmed that knockout of SNHG1 increased the mRNA and protein expressions of three migration markers, L1CAM, EGFR and PRKCA, and promote cell migration using cell migration assay, RT-qPCR and western blot. These results suggest that SNHG1 participates in a feedback loop regulating MYC and MYCN and promoted cell migration that contributes to neuroblastoma tumorigenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70408 |
DOI: | 10.6342/NTU201803210 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-1.pdf 目前未授權公開取用 | 4.52 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。