A MYC INDUCED LONG NON-CODING RNA, DANCR, THAT AFFECTS CELL PROLIFERATION

MYC;long non-coding RNA;Human Genetics and Molecular Biology

Lu, YunqiIsaacs, John ;

Johns Hopkins University

关键词: MYC;    long non-coding RNA;    Human Genetics and Molecular Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60505/LU-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Long non-coding RNAs (lncRNAs) are pervasively transcribed in the human genome and are known to be functioning in various cellular processes; however, the exact mechanisms involved are not well understood. The MYC proto-oncogene encodes a transcription factor that regulates cellular functions through the activation of its many RNA targets. In this thesis, I address the role of MYC-regulated lncRNAs in cancer.I first utilized experimental methods, such as microarray, RNA-seq, and TCGA analysis, to globally explore the regulatory role of MYC on lncRNAs. One lncRNA, DANCR, was identified as one of the most highly MYC-induced targets in human cancers. To further confirm that DANCR is directly regulated by MYC, I demonstrated by CHIP and CHIP-seq that MYC could bind directly to an E-box motif (CACGTG) in the DANCR promoter sequence. Using existing data and my own experimental analysis, DANCR expression was shown to be upregulated in most primary cancers. I next sought to determine whether DANCR is required for MYC-mediated cellular functions in cancer. Abrogation of DANCR expression using siRNA resulted in defective cell proliferation. Cell cycle analysis showed that the defect triggered by DANCR depletion was a consequence of G1 to S phase transition arrest. To assess the expression of genes responsible for this phenotype, I examined the expression profiles of cancer cells treated with DANCR siRNA. Gene Set Enrichment Analysis (GSEA) showed that a large subset of upregulated genes are common to DANCR- and EZH2-depletion. Moreover, p21, an important G1 phase cell cycle regulator, was found to be among those genes. I later showed that p21 could be suppressed by both DANCR and EZH2. More importantly, a double knock-down experiment of DANCR and p21 resulted in the partial rescue of the proliferation reduction caused by DANCR knock-down alone, providing evidence that p21 is one of the most important targets of DANCR.I proposed that MYC induces DANCR, which suppresses p21 via the EZH2 pathway, and then provided evidence for this model in a series of IP (immunoprecipitation) experiments to establish interactions between pathway components. A 3ʹ-domain of DANCR was shown to bind EZH2, whereas a 5ʹ-domain of DANCR recognized p21 nascent mRNA via RNA–RNA interactions, which guides DANCR–EZH2 complexes to the p21 locus. Finally, using computational approaches, I also attempted to explore whether my proposed mechanism could be generalized to other DANCR targets, and 8 out of 16 genes tested seemed to conform to this model. My findings elucidate an important missing ;;link’ in the MYC regulated gene network, showing active roles for lncRNAs in MYC-mediated cancer, which could be important targets for cancer diagnosis and therapy.Advisor: Dr. Chi DangReaders: Drs. Stephen Baylin and Chi DangOther Committee Members: Drs. John Isaacs and James Herman