- 學位論文
探討長鍊非編碼核糖核酸SNHG1在人類神經母細胞瘤的角色
Long Non-coding RNA SNHG1 in Neuroblastoma
摘要
神經母細胞瘤（neuroblastoma) 是一種好發於兒童的癌症，並且在臨床上被診斷出有不同的表徵及惡化程度，已知跟基因的突變機制有關並且會導致疾病的產生，其中之一為MYCN轉錄基因的異常放大。長鏈非編碼核糖核酸為長於200鹼基對類似訊息核糖核酸，但卻不會轉譯出蛋白質，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。由於長鏈非編碼核糖核酸 （long non-coding RNA）和MYCN轉錄基因與惡性神經母細胞瘤之間的關係仍不明確。在本研究中，我們結合核糖核酸測序（RNA-sequencing）與微陣列（Microarray）資料庫的基因表現調控網路，找到在神經母細胞瘤當中由MYCN致癌轉錄因子所調控的長鏈非編碼核糖核酸基因。我們發現small nucleolar RNA host gene 1 (SNHG1) 的表現量差異跟MYCN轉錄基因的異常放大表現量呈現高度的正相關；同時亦發現SNHG1表現較高的病人具有較差的預後。另一方面，我們利用即時聚合酶鏈式反應（qRT−PCR）證明SNHG1的表現量在MYCN異常放大神經母細胞瘤細胞株與MYCN非異常放大神經母細胞瘤細胞株具有表現量差異性。此外，在MYCN異常放大神經母細胞瘤細胞株中抑制MYCN的表現量後，SNHG1的表現量也隨之下降。SNHG1表現愈高預後越差。因此我們為了了解SNHG1的功能，利用核糖核酸蛋白質拉下測定實驗 （RNA-protein pull down assay）並結合液相層析串聯式質譜儀 （LC-MS/MS）測定蛋白身份，以得知與其交互作用的蛋白。最後，透過進一步的結合基序分析 （binding motif analysis）得知Matrin3, YBX1以及DDX5 可能會與SNHG1結合，並且利用西方點墨法（Western blot）證實Matrin 3會與SNHG1形成結合體。除此之外，我們進一步利用RNA免疫沉澱法也證實Matrin 3會與SNHG1直接結合。很多研究顯示Matrin 3是一種細胞骨架，我們猜測可能會與SNHG1形成結合體之後，參與調控神經母細胞瘤的細胞週期。本研究讓我們更深入瞭解SNHG1可能的功能，期望可以對神經母細胞瘤的治療發展有所貢獻。
並列摘要
Neuroblastoma (NB) is an embryonal tumor with various clinical presentations and behaviors. Several genomic alterations has been well-studied in NB, among which genomic amplification of MYCN oncogene, is a strong prognostic biomarker with worsens outcome. Long noncoding RNAs (lncRNAs), constitute major proportion of the cellular transcripts with no coding capacity. One of their function is to guide transcription factors to the target genes and facilitate gene expression. However, relative contribution of lncRNA and MYCN to the advanced NB has remained unclear. Herein, by applying a network-based integrative analysis on MYCN amplified and MYCN nonamplified lncRNA expression profile from both RNA-seq and microarray platform, we identified lncRNA, SNHG1 to be differentially expressed and strongly correlated with MYCN in MYCN-amplified NB. The expression of SNHG1 was validated by RT-qPCR in NB cell lines. Survival analysis revealed that higher expression of SNHG1 significantly associates with poor patient survival. Moreover, knockdown of MYCN in MYCN-amplified NB cell lines inhibited SNHG1 expression. Furthermore, to unravel the role of SNHG1 in NB, we extracted SNHG1-interacting proteins by RNA-protein pull down assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 27 SNHG1-interacting proteins in common from three NB cell lines. However, only three SNHG1-interacting proteins, MATR3, YBX1 and HHRNPL have binding site detected by DeepBind motif analysis. Western blot confirms interaction of MATR3 with SNHG1. Additionally, we further validated the direct interaction between MATR3 and SNHG1 by RNA-immunoprecipation (IP). MATR3 is known to be involved in RNA transport and stabilization. Therefore, we proposed that MATR3 after interacting with SNHG1 might help in SNHG1 transcription and stabilization. In conclusion, our study unveils that SNHG1 could be a prognostic marker for high-risk NB and possibly stabilized by MATR3. Our results might provide future directions for the development of therapeutic strategies against high-risk NB.
並列關鍵字
Neuroblastoma ； MYCN ； Long non-coding RNA ； SNHG1 ； RNA-protein pull down assay ； LC-MS/MS ； RNA-immunoprecipation (IP) ； MATR3參考文獻
延伸閱讀
- Lin, S. H. (2018). 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 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201803210
- Chen, Y. P. (2016). 探討在神經母細胞瘤中長鏈非編碼核糖核酸和MYCN調控網路的交互作用 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201601739
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