Molecular mechanism of chemoresistance by miR-215 in osteosarcoma and colon cancer cells.
BACKGROUND
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. In this study, we investigated the roles of miR-215 in the chemoresistance to DHFR inhibitor methotrexate (MTX) and TS inhibitor Tomudex (TDX).


RESULTS
The 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.


CONCLUSIONS
Taken 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.
Song
Bo
B
Translational Research Laboratory, Department of Pathology, School of Medicine, Stony Brook University, Stony Brook, New York 11794, USA.
Wang
Yuan
Y
Titmus
Matthew A
MA
Botchkina
Galina
G
Formentini
Andrea
A
Kornmann
Marko
M
Ju
Jingfang
J
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D044767:Ubiquitin-Protein Ligases
D000970:Antineoplastic Agents; C429530:DTL protein, human; D035683:MicroRNAs; D009687:Nuclear Proteins; D011799:Quinazolines; D034741:RNA, Small Interfering; D013876:Thiophenes; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D044767:Ubiquitin-Protein Ligases; C068874:raltitrexed; D008727:Methotrexate