Experimental verification of a predicted intronic microRNA in human NGFR gene with a potential pro-apoptotic function

PLoS One. 2012;7(4):e35561. doi: 10.1371/journal.pone.0035561. Epub 2012 Apr 27.

Abstract

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells.

MeSH terms

  • Apoptosis / genetics
  • Base Sequence
  • Cell Line, Tumor
  • Exons / genetics
  • Gene Expression Regulation, Neoplastic*
  • Glioma / genetics
  • Glioma / pathology
  • HeLa Cells
  • Humans
  • Introns / genetics*
  • Lipoprotein Lipase / genetics
  • Lipoprotein Lipase / metabolism
  • MicroRNAs / chemistry
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Molecular Sequence Data
  • Nerve Growth Factors / metabolism
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Plasmids
  • Receptors, Nerve Growth Factor / genetics*
  • Receptors, Nerve Growth Factor / metabolism
  • Signal Transduction / genetics
  • TRPP Cation Channels / genetics
  • TRPP Cation Channels / metabolism
  • Transfection

Substances

  • MIRN6165 microRNA, human
  • MicroRNAs
  • NGFR protein, human
  • Nerve Growth Factors
  • Nerve Tissue Proteins
  • Receptors, Nerve Growth Factor
  • TRPP Cation Channels
  • polycystic kidney disease 1 protein
  • DAGLA protein, human
  • Lipoprotein Lipase

Associated data

  • GENBANK/FR873488