title: Role of heme oxygenase-1 in demethylating effects on SKM-1 cells induced by decitabine.
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.
authors.affiliation: Department of Hematology, Affiliated Hospital of Guiyang Medical University, Guiyang, China.|Key Laboratory of Hematological Disease Diagnostic & Treat Centre of Guizhou Province, Guiyang, China.|Guizhou Province Hematopoietic Stem Cell Transplantation Center, Affiliated Hospital of Guiyang Medical University, Guiyang, China.
authors.forename: R
authors.initials: R
authors.lastname: Gao
authors.affiliation: Department of Hematology, Affiliated Hospital of Guiyang Medical University, Guiyang, China.|Key Laboratory of Hematological Disease Diagnostic & Treat Centre of Guizhou Province, Guiyang, China.|Guizhou Province Hematopoietic Stem Cell Transplantation Center, Affiliated Hospital of Guiyang Medical University, Guiyang, China.|Department of Pharmacy, Affiliated Baiyun Hospital of Guiyang Medical University, Guiyang, China.
authors.forename: D
authors.initials: D
authors.lastname: Ma
authors.affiliation: Department of Hematology, Affiliated Hospital of Guiyang Medical University, Guiyang, China.|Key Laboratory of Hematological Disease Diagnostic & Treat Centre of Guizhou Province, Guiyang, China.|Guizhou Province Hematopoietic Stem Cell Transplantation Center, Affiliated Hospital of Guiyang Medical University, Guiyang, China.
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.affiliation: School of Pharmacy, Guiyang Medical University, Guiyang, China.
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Hematology, Affiliated Hospital of Guiyang Medical University, Guiyang, China.|Key Laboratory of Hematological Disease Diagnostic & Treat Centre of Guizhou Province, Guiyang, China.|Guizhou Province Hematopoietic Stem Cell Transplantation Center, Affiliated Hospital of Guiyang Medical University, Guiyang, China.
authors.forename: J S
authors.initials: JS
authors.lastname: Wang
authors.affiliation: Department of Pharmacy, Affiliated Baiyun Hospital of Guiyang Medical University, Guiyang, China.|Department of Pharmacy, Affiliated Hospital of Guiyang Medical University, Guiyang, China.
authors.forename: Q
authors.initials: Q
authors.lastname: Fang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000096:biosynthesis / Q000235:genetics*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis* / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic; D051547:Heme Oxygenase-1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: C493966:CDKN2B protein, human; D050762:Cyclin-Dependent Kinase Inhibitor p15; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D000077209:Decitabine; D051547:Heme Oxygenase-1; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D001374:Azacitidine