Loss-of-function screening to identify miRNAs involved in senescence: tumor suppressor activity of miRNA-335 and its new target CARF

Sci Rep. 2016 Jul 26:6:30185. doi: 10.1038/srep30185.

Abstract

Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs, and coupled it with a cell based loss-of-function (5-Aza-2'-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16(INK4A), pRB, HDM2 and p21(WAF1).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / metabolism*
  • Azacitidine / analogs & derivatives
  • Azacitidine / pharmacology
  • Cell Line, Tumor
  • Cellular Senescence / drug effects
  • Decitabine
  • Genes, Tumor Suppressor*
  • Genes, p16
  • Heterografts
  • Humans
  • Mice
  • Mice, Nude
  • MicroRNAs / genetics
  • MicroRNAs / physiology*
  • RNA-Binding Proteins / metabolism*
  • Retinoblastoma Protein / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • CDKN2AIP protein, human
  • MIRN335 microRNA, human
  • MicroRNAs
  • RNA-Binding Proteins
  • Retinoblastoma Protein
  • Decitabine
  • Azacitidine