ABSTRACT
MicroRNAs are predicted to regulate approximately 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.
Subject(s)
MicroRNAs/metabolism , Polymorphism, Single Nucleotide , Tetrahydrofolate Dehydrogenase/genetics , Animals , Base Sequence , Binding Sites , CHO Cells , Cricetinae , Cricetulus , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA, Messenger/geneticsABSTRACT
Pemetrexed was approved for the treatment of relapsed or chemotherapy refractory non-small cell lung cancer patients, as it produced similar response and survival outcomes and less toxicity as compared to taxotere. Pemetrexed in combination with platinum analogs or with gemcitabine or vinorelbine, produce equivalent responses and overall survival results compared to combinations of platinum analogs with other drugs. The role of bevacizumab and the inhibitors of epithelial growth factor receptor also should be evaluated in selected patients with NSCLC treated with pemetrexed combinations. Further increases in drug dose may be possible using transfer of drug resistance genes in hematopoietic stem cells.
