Abstract
Dihydrofolate reductase (DHFR) plays a key role in folate metabolism and is a target molecule of methotrexate. An increase in the cellular expression level of DHFR is one of the mechanisms of tumor resistance to methotrexate. The present study investigated the possibility that adenosine-to-inosine RNA editing, which causes nucleotide conversion by adenosine deaminase acting on RNA (ADAR) enzymes, might modulate DHFR expression. In human breast adenocarcinoma-derived MCF-7 cells, 26 RNA editing sites were identified in the 3'-UTR of DHFR. Knockdown of ADAR1 decreased the RNA editing levels of DHFR and resulted in a decrease in the DHFR mRNA and protein levels, indicating that ADAR1 up-regulates DHFR expression. Using a computational analysis, miR-25-3p and miR-125a-3p were predicted to bind to the non-edited 3'-UTR of DHFR but not to the edited sequence. The decrease in DHFR expression by the knockdown of ADAR1 was restored by transfection of antisense oligonucleotides for these miRNAs, suggesting that RNA editing mediated up-regulation of DHFR requires the function of these miRNAs. Interestingly, we observed that the knockdown of ADAR1 decreased cell viability and increased the sensitivity of MCF-7 cells to methotrexate. ADAR1 expression levels and the RNA editing levels in the 3'-UTR of DHFR in breast cancer tissues were higher than those in adjacent normal tissues. Collectively, the present study demonstrated that ADAR1 positively regulates the expression of DHFR by editing the miR-25-3p and miR-125a-3p binding sites in the 3'-UTR of DHFR, enhancing cellular proliferation and resistance to methotrexate.
Highlights
Thesis required for DNA synthesis, cell growth, and proliferation, Dihydrofolate reductase (DHFR) is a target of chemotherapeutic agents such as methotrexate and pemetrexed [1,2,3]
A significant decreased in DHFR protein levels by siADAR1 was observed (Fig. 1H)
By selecting 3 samples of which ADAR1 levels in cancer tissue were higher than in normal tissues (ADAR1 expression tumor/normal ratios of 9.7, 14.0, and 16.7) and 3 samples of which ADAR1 levels in cancer tissues were equal to or lower than in normal tissues (0.1, 0.9, and 1.0), the RNA editing levels in the 3Ј-UTR of DHFR were evaluated. In the former samples, the RNA editing levels of the DHFR transcript were higher in cancer tissues than in adjacent normal tissues (Fig. 6B), but in the latter samples (Fig. 6C), this phenomenon was not observed. These results suggest that the increased expression of ADAR1 in breast cancer contributes to hyper-editing of the DHFR transcript, which would result in an increase in DHFR expression
Summary
Exons, changing crucial amino acids for protein function. Accumulating evidence has revealed that aberrant ADAR expression and disrupted RNA editing levels are associated with diseases including cancer, metabolic diseases, viral infections, autoimmune disorders, and neurological disorders [34]. Multiple RNA editing sites were identified at introns 3, 4, and 5 as well as 3Ј-UTR of DHFR mRNA in the lymphoblastoid cell line and brain [37, 38]. It is uncertain whether the DHFR is edited or not in breast cancer, in which ADAR1 has been reported to function as an oncogene [39]. We investigated the possibility that RNA editing might modulate DHFR expression and subsequently affect the proliferation and sensitivity of breast cancer cells to methotrexate
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
