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

doi:10.1073/pnas.0706217104

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

*Department of Pharmacology, Graduate School of Biomedical Sciences, and
^†Department of Pharmacology and Medicine, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903

Communicated by Allan H. Conney, Rutgers, The State University of New Jersey, Piscataway, NJ, July 2, 2007 (received for review May 7, 2007)

Abstract

MicroRNAs are predicted to regulate ≈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→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→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→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.

Footnotes

^§To whom correspondence should be addressed at:
Department of Medicine and Pharmacology, Cancer Institute of New Jersey, Room 3034, 195 Little Albany Street, New Brunswick, NJ 08903.
E-mail: bertinoj{at}umdnj.edu
Author contributions: Prasun J. Mishra, D.B., and J.R.B. designed research; Prasun J. Mishra, R.H., Pravin J. Mishra, and G.S.A.L.-S. performed research; Prasun J. Mishra analyzed data; and Prasun J. Mishra, D.B., and J.R.B. wrote the paper.
↵ ^‡Present address: Department of Hematology, Centro di Riferimento Oncologico, 98039 Taormina, Italy.
The authors declare no conflict of interest.
Abbreviations:

MTX,

methotrexate;

miRNA,

microRNA;

RISC,

RNA-induced silencing complex;

DHFR,

dihydrofolate reductase;

Ago,

Argonaute.