Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer

PLoS One. 2014 Jun 3;9(6):e96767. doi: 10.1371/journal.pone.0096767. eCollection 2014.

Abstract

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Animals
  • Apoptosis / genetics*
  • Cell Proliferation
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / pathology*
  • DNA Methylation / genetics
  • Down-Regulation / genetics
  • Female
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • Gene Knockdown Techniques
  • HCT116 Cells
  • High-Throughput Screening Assays*
  • Humans
  • Intestinal Mucosa / pathology
  • Laser Capture Microdissection
  • Male
  • Mice
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Middle Aged
  • Neoplasm Proteins / metabolism
  • Neoplasm Staging
  • Phenotype
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism
  • Reproducibility of Results

Substances

  • MicroRNAs
  • Neoplasm Proteins
  • RNA, Messenger
  • RNA, Small Interfering

Grant support

This work was supported by the Danish National Advanced Technology Foundation, the John and Birthe Meyer Foundation, the Danish Council for Independent Research (Medical Sciences), the Danish Council for Strategic Research, the Lundbeck Foundation and The European Union's Seventh Framework Program (SYSCOL HEALTH-F5-2010-258236). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.