Journal Article

miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance

Banerjee, Debabrata, Mishra, Prasun J, Humeniuk, Rita, Bertino, Joseph R, Longo-Sorbello, Giuseppe S.A, Mishra, Pravin J

2007

Overview

MicroRNAs are predicted to regulate [almost equal to]30% of all human genes by targeting sequences in their 3' UTR. Polymorphisms in 3' UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood. Here, we demonstrate that 829C[rightward arrow]T, a naturally occurring SNP, near the miR-24 binding site in the 3' UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance. miR-24 has a conserved binding site in DHFR 3' UTR. DHFR with WT and 3' UTR containing the 829C[rightward arrow]T mutation were expressed in DG44 cells that lack DHFR. Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3' UTR-expressing cells. Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells. Cells with the mutant 3' UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells. SNP-829C[rightward arrow]T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate. We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3' UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: 3' untranslated regions / Animals / Base Sequence / Binding Sites / Biological Sciences / Cell lines / CHO Cells / Cricetinae / Cricetulus / dihydrofolate reductase / DNA / Drug resistance / Gene expression / Gene expression regulation / gene overexpression / genes / Genetic mutation / half life / humans / Messenger RNA / methotrexate / MicroRNA / MicroRNAs - metabolism / Molecular Sequence Data / Mutagenesis, Site-Directed / mutants / Mutation / Polymorphism / Polymorphism, Single Nucleotide / protein synthesis / Ribonucleic acid / RNA / RNA, Messenger - genetics / single nucleotide polymorphism / Tetrahydrofolate Dehydrogenase - genetics / Untranslated regions