Research Article

Role of heme oxygenase-1 in demethylating effects on SKM-1 cells induced by decitabine

Published: December 22, 2015
Genet. Mol. Res. 14 (4) : 17788-17798 DOI: 10.4238/2015.December.22.3

Abstract

We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.

We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.