Regulation of receptor tyrosine kinases by miRNA: overexpression of miRNA using lentiviral inducible expression vectors

Methods Mol Biol. 2015;1233:135-47. doi: 10.1007/978-1-4939-1789-1_13.

Abstract

MicroRNAs have the ability to alter and regulate multiple genes, including RTK family members, making them an attractive approach for molecular therapeutic development. We use a pCDNA6.2-EmGFP-microRNA expression vector to overexpress individual mature microRNA and then transfer the expression cassette into a single, inducible lentiviral vector (pINDUCER20). We successfully use this system to create a pINDUCER-EmGFP-miRNA27a expression vector and generate a stable head and neck cancer cell line (UM-SCC-22A) that inducibly expresses miRNA-27a, resulting in targeted epidermal growth factor receptor down regulation. In this chapter, we describe the protocol for engineering the pINDUCER-EmGFP-microRNA expression vector, producing lentiviral particles for target cell infection, and evaluating downregulation of gene expression.

MeSH terms

  • Cell Line, Tumor
  • ErbB Receptors / genetics*
  • ErbB Receptors / metabolism
  • Gene Expression Regulation, Neoplastic*
  • Genes, Reporter
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Lentivirus / genetics*
  • Lentivirus / metabolism
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Oligonucleotides / chemical synthesis
  • Oligonucleotides / metabolism
  • Plasmids / chemistry*
  • Plasmids / metabolism
  • Signal Transduction
  • Transfection
  • Virion / genetics*
  • Virion / metabolism

Substances

  • MIRN27 microRNA, human
  • MicroRNAs
  • Oligonucleotides
  • Green Fluorescent Proteins
  • EGFR protein, human
  • ErbB Receptors