The Study On The Enhancing Effects Of HO-1 Silencing On 5-azacytidine-induced Apoptosis Of SKM-1 Cells

Objective: To explore the enhancing effects and mechanisms of interfering RNA-mediated HO-1 silencing on apoptosis induced by low concentration of AZA in SKM-1 cells.Methods: Patient bone marrow mononuclear cells(BMNCs)(n=48) diagnosed as different levels of MDS were collected. HO-1 mRNA were deteced in MDS/AML cell lines with AZA treatment or not and in MDS-derived BMNCs by real-time PCR. SKM-1 cells were transfected with lentivirus-mediated interfering RNA to silence HO-1 expression, and further grouped into three groups: blank group, empty vector group and HO-1 silencing group. Hemin was used to upregulate HO-1 expression. Cell growth was evaluated by MTT assay, cell cycle and apoptosis were detected by flow cytometry. Next, DNMT1, p16 and its downstream cell cycle related genes(CDK4, CDK6, CyclinD1, p21, p27, p57, RB and E2F1) were analyzed after HO-1 regulation by real-time PCR, these cell cycle-related proteins and apoptosis-related proteins(caspase-3,cleaved caspase- 3, BCL-2 and Bax) were further examined through western blot analysis.Methylation was assessed by MSP(methylation-specific PCR). The intravenous MDS mouse model was built in NOD/SCID mouse, weight loss and survival time of the mouse were recorded,the status of injected SKM-1 cells in peripheral blood was confirmed by Wright staining, SKM-1cells were further tested by flow cytometry in peripheral blood. Results: HO-1 overexpression was observed in SKM-1 cells after AZA treatment comparing to other cell lines. The HO-1expression levels of high-risk and very-high-risk MDS patients exceeded those of low-risk and very-low-risk patients. After HO-1 was silenced by lentivirus-mediated siRNA, the proliferation of SKM-1 cells was effectively inhibited by low concentration AZA, in contrast, this proliferation inhibition was blunted when Hemin was used. Flow cytometry assay showed that the cells were more prone to apoptosis and arrested in the G0/G1 phase, whereas, these effects were weakened after hemin treatment. Upregulation of p16 and changes of p16-relative cell cycle and apoptosis-related proteins were observed after silencing HO-1 in AZA treated SKM-1 cells. In addition, DNMT1 was downregulated following the decrease of HO-1 expression. After HO-1silencing, the promoter region of p16 gene was further demethylated after AZA exposure, however,the demethyaltion of p16 gene promoter was reduced with Hemin exposure. In vivo, silencing HO-1 further inhibited SKM-1 cell growth induced by AZA in an intravenous MDS NOD/SCID mouse model, with decreased weight loss, prolonged survival time and reduced CD45+ SKM-1cells in peripheral blood. Conclusion: Silencing HO-1 sensitized SKM-1 cells toward AZA,which may be attributed to the influence of silencing HO-1 on promoting AZA-induced p16 gene demethylation, meanwhile, the expressions of cleaved caspase-3 and Bax increased, whereas that of BCL-2 decreased. HO-1 may be one of the targets that enhance the therapeutic effects of AZA on MDS malignant transformation, which inspires new treatment methods for high-risk MDS patients in clinical practice.