Role of MYC-regulated long noncoding RNAs in cell cycle regulation and tumorigenesis.

BACKGROUND The functions of long noncoding RNAs (lncRNAs) have been identified in several cancers, but the roles of lncRNAs in colorectal cancer (CRC) are less well understood. The transcription factor MYC is known to regulate lncRNAs and has been implicated in cancer cell proliferation and tumorigenesis. METHODS CRC cells and tissues were profiled to identify lncRNAs differentially expressed in CRC, from which we further selected MYC-regulated lncRNAs. We used luciferase promoter assay, ChIP, RNA pull-down assay, deletion mapping assay, LC-MS/MS and RNA immunoprecipitation to determine the mechanisms of MYC regulation of lncRNAs. Moreover, soft agar assay and in vivo xenograft experiments (four athymic nude mice per group) provided evidence of MYC-regulated lncRNAs in cancer cell transformation and tumorigenesis. The Kaplan-Meier method was used for survival analyses. All statistical tests were two-sided. RESULTS We identified lncRNAs differentially expressed in CRC (P < .05, greater than two-fold) and verified four lncRNAs upregulated and two downregulated in CRC cells and tissues. We further identified MYC-regulated lncRNAs, named MYCLos. The MYC-regulated MYCLos may function in cell proliferation and cell cycle by regulating MYC target genes such as CDKN1A (p21) and CDKN2B (p15), suggesting new regulatory mechanisms of MYC-repressed target genes through lncRNAs. RNA binding proteins including HuR and hnRNPK are involved in the function of MYCLos by interacting with MYCLo-1 and MYCLo-2, respectively. Knockdown experiments also showed that MYCLo-2, differentially expressed not only in CRC but also in prostate cancer, has a role in cancer transformation and tumorigenesis. CONCLUSIONS Our results provide novel regulatory mechanisms in MYC function through lncRNAs and new potential lncRNA targets of CRC.

[1]  R. Johnstone,et al.  The role of p21waf1/cip1 and p27Kip1 in HDACi-mediated tumor cell death and cell cycle arrest in the Eμ-myc model of B-cell lymphoma , 2014, Oncogene.

[2]  Howard Y. Chang,et al.  Dicer-microRNA-Myc circuit promotes transcription of hundreds of long noncoding RNAs , 2014, Nature Structural &Molecular Biology.

[3]  C. Croce,et al.  Long-range interaction and correlation between MYC enhancer and oncogenic long noncoding RNA CARLo-5 , 2014, Proceedings of the National Academy of Sciences.

[4]  J. Foekens,et al.  CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer , 2013, Genome research.

[5]  Tak W. Mak,et al.  Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15. , 2013, Genes & development.

[6]  Jun Wang,et al.  Multiple Functions of the RNA-Binding Protein HuR in Cancer Progression, Treatment Responses and Prognosis , 2013, International journal of molecular sciences.

[7]  Nashi Widodo,et al.  Heterogeneous Nuclear Ribonucleoprotein K (hnRNP-K) Promotes Tumor Metastasis by Induction of Genes Involved in Extracellular Matrix, Cell Movement, and Angiogenesis , 2013, The Journal of Biological Chemistry.

[8]  Steven J. M. Jones,et al.  Comprehensive molecular characterization of human colon and rectal cancer , 2012, Nature.

[9]  Q. Zhan,et al.  BAG2 is a target of the c-Myc gene and is involved in cellular senescence via the p21(CIP1) pathway. , 2012, Cancer letters.

[10]  Gerd Ritter,et al.  Colon cancer associated transcript‐1: A novel RNA expressed in malignant and pre‐malignant human tissues , 2012, International journal of cancer.

[11]  Chi V Dang,et al.  MYC on the Path to Cancer , 2012, Cell.

[12]  J. Rinn,et al.  Modular regulatory principles of large non-coding RNAs , 2012, Nature.

[13]  D. Dobbs,et al.  Predicting RNA-Protein Interactions Using Only Sequence Information , 2011, BMC Bioinformatics.

[14]  T. Derrien,et al.  Long Noncoding RNAs with Enhancer-like Function in Human Cells , 2010, Cell.

[15]  P. Fortina,et al.  p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo , 2009, Proceedings of the National Academy of Sciences.

[16]  J. Rinn,et al.  Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression , 2009, Proceedings of the National Academy of Sciences.

[17]  Thomas D. Schmittgen,et al.  Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. , 2007, Cancer cell.

[18]  Howard Y. Chang,et al.  Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs , 2007, Cell.

[19]  G I Murray,et al.  Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer , 2006, British Journal of Cancer.

[20]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[21]  A. Trumpp,et al.  Skin epidermis lacking the c-Myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene. , 2006, Genes & development.

[22]  J. Mattick,et al.  Small regulatory RNAs in mammals. , 2005, Human molecular genetics.

[23]  D. Beach,et al.  Absence of p21WAF1 cooperates with c-myc in bypassing Ras-induced senescence and enhances oncogenic cooperation , 2004, Oncogene.

[24]  J. Massagué,et al.  TGFβ influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b , 2001, Nature Cell Biology.

[25]  J. Massagué,et al.  Repression of p15INK4b expression by Myc through association with Miz-1 , 2001, Nature Cell Biology.

[26]  R. Herrmann,et al.  Overexpression and amplification of c-myc during progression of human colorectal cancer. , 1996, Oncology.

[27]  Ewan Birney,et al.  Cell-type specific and combinatorial usage of diverse transcription factors revealed by genome-wide binding studies in multiple human cells. , 2012, Genome research.