Abstract
BackgroundTranslational control mediated by non-coding microRNAs (miRNAs) plays a key role in the mechanism of cellular resistance to anti-cancer drug treatment. Dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS, TS) are two of the most important targets for antifolate- and fluoropyrimidine-based chemotherapies in the past 50 years. In this study, we investigated the roles of miR-215 in the chemoresistance to DHFR inhibitor methotrexate (MTX) and TS inhibitor Tomudex (TDX).ResultsThe protein levels of both DHFR and TS were suppressed by miR-215 without the alteration of the target mRNA transcript levels. Interestingly, despite the down-regulation of DHFR and TS proteins, ectopic expression of miR-215 resulted in a decreased sensitivity to MTX and TDX. Paradoxically, gene-specific small-interfering RNAs (siRNAs) against DHFR or TS had the opposite effect, increasing sensitivity to MTX and TDX. Further studies revealed that over-expression of miR-215 inhibited cell proliferation and triggered cell cycle arrest at G2 phase, and that this effect was accompanied by a p53-dependent up-regulation of p21. The inhibitory effect on cell proliferation was more pronounced in cell lines containing wild-type p53, but was not seen in cells transfected with siRNAs against DHFR or TS. Moreover, denticleless protein homolog (DTL), a cell cycle-regulated nuclear and centrosome protein, was confirmed to be one of the critical targets of miR-215, and knock-down of DTL by siRNA resulted in enhanced G2-arrest, p53 and p21 induction, and reduced cell proliferation. Additionally, cells subjected to siRNA against DTL exhibited increased chemoresistance to MTX and TDX. Endogenous miR-215 was elevated about 3-fold in CD133+HI/CD44+HI colon cancer stem cells that exhibit slow proliferating rate and chemoresistance compared to control bulk CD133+/CD44+ colon cancer cells.ConclusionsTaken together, our results indicate that miR-215, through the suppression of DTL expression, induces a decreased cell proliferation by causing G2-arrest, thereby leading to an increase in chemoresistance to MTX and TDX. The findings of this study suggest that miR-215 may play a significant role in the mechanism of tumor chemoresistance and it may have a unique potential as a novel biomarker candidate.
Highlights
Translational control mediated by non-coding microRNAs plays a key role in the mechanism of cellular resistance to anti-cancer drug treatment
Dihydrofolate reductase (DHFR) and TS are direct targets of miR-215 Given the significance of DHFR and TS as two of the major targets of anti-cancer chemotherapy, we used a bioinformatics approach to identify miRNAs that were predicted to bind to DHFR and TS mRNAs and function in their regulation
Using TargetScan and PicTar in the miRNAs database, we found that miR-215 was predicted to have a potential interaction site at the 3'UTR of DHFR mRNA and two sites at the 3'UTR of the TS mRNA (Figure 1A)
Summary
Translational control mediated by non-coding microRNAs (miRNAs) plays a key role in the mechanism of cellular resistance to anti-cancer drug treatment. Dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS, TS) are two of the most important targets for antifolate- and fluoropyrimidine-based chemotherapies in the past 50 years. It has been well documented that post-transcriptional and translational controls play a key role in the mechanism of cellular resistance to anti-cancer drug treatment [7,8,9,10,11,12]. A given species of miRNA can perfectly or imperfectly base pair with multiple targets, allowing it to potentially regulate the translation of numerous mRNAs. It has been predicted that over 30% of the human protein coding genes are post-transcriptionally regulated by this mechanism [16,17,18]. Given the major roles of miRNAs in the regulation of protein expression in general, it is crucial to understand the contributions of miRNAs to tumor chemoresistance
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