Expression Of Nucleoside-metabolizing Enzymes In Myelodysplastic Syndrome And Mechanism To Resistance To Decitabine

Objective: This study was aim to investigate expression levels of genes involved in decitabine metabolism using quantitative PCR in myelodysplastic syndrome(MDS) who received decitabine. And explore the mechanism in resistance to decitabine.Methods:We determined the expression levels of genes involved in decitabine metabolism: human equilibrative nucleoside transporter 1(h ENT1), deoxycytidine kinase(DCK) and cytidine deaminase(CDA) using quantitative PCR in samples from 34 patients with MDS who received decitabine. h ENT1 was silenced in MDS derived cell line SKM-1 mediated by lentivirus transfection. The infection efficiency was detected by flow cytometry, and the m RNA expression level of h ENT1 was confirmed by q RT-PCR. At 24 h,48h and 72 h, the proliferation ratio of SKM-1 treated with decitabine(0.5?1?5 ?mol/L) was detected by CCK-8 method after silencing the h ENT1, and the apoptosis of SKM-1 was detected by Western blot for cleaved level of caspase-3, demethylation of p15INK4 B induced by decitabine in SKM-1 were also analyzed.Results: In this study, we confirmed that h ENT1 m RNA expression levels was significantly higher in 18 response patients compared with 16 non-response patients(2.95±3.12 vs 1.23±0.73; p=0.035), Which is an important equilibrative nucleoside transporter that transport decitabine into cells; DCK and CDA had no significant difference bettween the two groups. We also found that the MDS patients whose hENT1 m RNA expression above the h ENT1 median levels had longer overall survival(OS) than the patients whose h ENT1 below the median levels. After silencing h ENT1 in SKM-1 cell line, we selected 3 concentrations of decitabine(0.5, 1, 5?mol/L) to treat SKM-1 KD and SKM-1 Ctrl at 24 h, 48 h and 72 h. We found that the proliferation activity of SKM-1 KD was not significantly impaired(p<0.05),especially at the point of 72 h with 5?mol/L decitabine,the proliferation inhibition rate of Ctrl and KD were(49.41±4.02)% vs(33.03±2.47)%(p=0.007); Western blot showed that cleaved caspase3 of SKM-1 KD and the p15INK4 B demethylation status were significantly decreased in comparison with SKM-1 Ctrl(p<0.05).Conclusion: Taken together, our results suggest that the expression of h ENT1 is correlated with clinical outcome and may influence the clinical response to decitabine treatment in patients with MDS. Silencing of h ENT1 by si RNA leads to blunted response to decitabine in vitro. h ENT1 may play an important role in the resistance to decitabine.