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Published on: December 9, 2015
[Functional study of hENT1 on SKM-1 cell resistance to decitabine]
Wenhui Shi1, Lingyun Wu1, Juan Guo1
1Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
Objective:
To investigate the effect of human equilibrative nucleoside transporters 1 (hENT1) silencing on proliferation, apoptosis and demethylation of human myelodysplastic syndrome (MDS) derived cell line SKM-1 treated with 5-aza-2'-deoxycytidine (decitabine, DAC).
Methods:
hENT1 was silenced in SKM-1 cells mediated by lentivirus transfection. The infection efficiency was detected by flow cytometry, and the mRNA expression level of hENT1 was confirmed by qRT-PCR. The proliferation ratio of SKM-1 cells treated with different concentrations (0.5, 1, 5 mmol/L) of DAC for 24, 48 and 72 h was detected by CCK-8 method after hENT1 silencing. The apoptosis of SKM-1 cells was detected by Western blot for cleaved level of caspase-3 and evaluated by flow cytometry after staining with anti-Annexin V-PE and 7-AAD. The p15(INK4B) DNA methylation status was measured by methylation specific PCR using EZ DNA Methylation-Gold™ Kit.
Results:
The expression level of hENT1 silenced group (0.253±0.030) was statistically decreased compared with that in control group (1.000±0.091) (P<0.01). Compared with control, the proliferation inhibition rate of hENT1 silenced group was significantly decreased by different concentrations of DAC (0.5, 1, 5 μmol/L) treatment for 24, 48, 72 h (P<0.05), which was (49.41±4.02)% and (33.03±2.47)%, respectively (P=0.007) at 5 μmol/L DAC treatment for 72 h in hENT1 silenced group and the control group. Western blot showed that cleaved caspase3 of hENT1 silenced group was also significantly inhibited. The percentage of Annexin Ⅴ⁺ cells and demethylation status of p15(INK4B) were significantly decreased.
Conclusion:
Apoptosis of hENT1 silenced SKM-1 cells induced by DAC was decreased, and the susceptibility of these cells to demethylation treatment was also decreased.
Insights
Silencing human equilibrative nucleoside transporter 1 (hENT1) in myelodysplastic syndrome cells reduces their response to decitabine treatment. This leads to decreased proliferation inhibition, apoptosis, and DNA demethylation.
Area of Science:
- Hematology
- Molecular Biology
- Cancer Research
Background:
- Human myelodysplastic syndrome (MDS) is a group of clonal hematopoietic stem cell disorders.
- Decitabine (DAC) is a hypomethylating agent used in MDS treatment.
- The role of human equilibrative nucleoside transporter 1 (hENT1) in DAC efficacy is not fully understood.
Purpose of the Study:
- To investigate the impact of hENT1 silencing on the proliferation, apoptosis, and demethylation of the SKM-1 MDS cell line treated with DAC.
- To elucidate the mechanism by which hENT1 influences DAC sensitivity in MDS cells.
Main Methods:
- hENT1 was silenced in SKM-1 cells using lentivirus transfection.
- Cell proliferation was assessed using the CCK-8 assay.
- Apoptosis was evaluated by Western blot for cleaved caspase-3 and flow cytometry (Annexin V/7-AAD staining).
- DNA methylation status of p15(INK4B) was analyzed by methylation-specific PCR.
Main Results:
- hENT1 silencing significantly reduced hENT1 mRNA expression.
- Silencing hENT1 decreased DAC-induced proliferation inhibition in SKM-1 cells.
- Reduced apoptosis (decreased cleaved caspase-3, lower Annexin V positive cells) was observed in hENT1-silenced cells.
- Demethylation of p15(INK4B) was significantly diminished following hENT1 silencing.
Conclusions:
- hENT1 plays a crucial role in mediating the effects of decitabine in MDS cells.
- Silencing hENT1 reduces the sensitivity of SKM-1 cells to DAC, impairing its anti-proliferative and pro-apoptotic effects.
- Targeting hENT1 may represent a strategy to modulate DAC efficacy in MDS treatment.
