The Effects And The Mechanisms Of Heme Oxygenase On Prognosis In Patients With Lower-Risk Myelodysplastic Syndromes

Background:Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem-cell diseases. A few oxidative stress factors derived from lower-risk MDS bone marrow microenvironment lead to increasing rate of apoptosis in hematopoietic stem and progenitor cells, resulting in ineffective hematopoiesis and peripheral blood cytopenia. The complications such as severe infection, hemorrhage and heart failure have influenced the time and quality of life in lower-risk MDS patients. It was broadly approved that inhibiting excessive apoptosis of bone marrow cells can effectively improve hematopoiesis and prolong the duration of survival, which proved to be a new pathway to ameliorate prognosis of lower-risk MDS patients. Heme oxygenase-1(HO-1), known as an anti-oxidative enzyme, can eliminate intracellular oxygen free radicals and decrease cell apoptosis by regulating the activity of mitogen-activated protein kinases (MAPKs) in a distinct cell type pattern. Recent studies have demonstrated that the pathogenesis of MDS, especially the regulation of bone marrow cell proliferation and apoptosis, is strongly related with dysregulation of MAPKs. And inhibition of dysregulated MAPKs can restore hematopoiesis. However it is still not clarified whether HO-1as the upstream molecule of MAPKs could regulate the activity of MAPKs in bone marrow cells of lower-risk MDS.Objective:This study aimed to investigating the relationship between basic expression level of HO-1protein and the prognosis of lower-risk MDS patients, and to exploring the role of HO-1in regulating MAPK signaling pathway in bone marrow mononuclear cells (BMMNCs) of lower-risk MDS. Our study will further clarify the possible mechanism that HO-1involved and may offer a novel therapeutic target for treatment of MDS.Methods:1. Patients and controls:bone marrow aspirates were obtained from70lower-risk MDS patients who were newly diagnosed, including58patients with refractory anemia (RA) and12patients with refractory anemia with ring sideroblasts (RARS). And20healthy volunteers were studied as control group.2. Sample intervention:BMMNCs from70lower-risk MDS patients and normal controls were separated by standard Ficoll-Hypaque density gradient sedimentation. We randomly chose20patients from the above total70patients and treated their BMMNCs with HO-1inducer Hemin at different final concentrations of5u M,10μ M,20μM for48hours.3. Real-time reverse-transcription polymerase chain reaction (RT-PCR):we detected the basic expression level of HO-1mRNA in70lower-risk MDS and normal controls. After treated with Hemin HO-1mRNA was detected in every experimental group.4. Western blot:we detected the basic expression level of HO-1protein in70lower-risk MDS and normal controls. The expression levels of HO-1protein, phosphorylated ERK (p-ERK), phosphorylated JNK (p-JNK) and phosphorylated p38MAPK (p-p38MAPK) were detected in every experimental group after treated with Hemin.5. All70patients completed follow-up. Progression-free survival (PFS) was calculated from the date of diagnosis until the progression of disease or until the date of final follow-up. The relationship between HO-1expression levels and duration of PFS were further analysed.6. Statistical analysis:All tests were performed with SPSS (version17.0), P<0.05was considered significant. Among more than two groups was determined by LSD-t test. The difference between two groups was determined by rank-sum test if the data were not normally distributed. PFS were plotted with the use of the Lifetest procedure for Kaplan-Meier estimates and log-rank test. Cox proportional regression model and Spearman rank correlation were performed.Results:1. The univariate analysis indicated that the median duration of PFS of patients with higher expression level of HO-1was significantly longer than that with lower expression level of HO-1(40.8months vs32.6months, P=0.027). Cox regression showed that HO-1was an independent prognostic factor (HR=0.206,95%CI0.064-0.659, P=0.008). Spearman rank correlation showed that the expression levels of HO-1protein exhibited a positive correlation with the levels of hemoglobin in peripheral blood (r=0.389, P<0.05).2. No significant difference in HO-1mRNA and protein expression was observed between the lower-risk MDS subgroup and the normal controls (P>0.05). After treating BMMNCs with different concentrations of hemin, the expression of HO-1mRNA was significantly increased at groups with hemin compared with groups without hemin in lower-risk MDS (P<0.05). There was an increase of HO-1mRNA and protein in normal controls, but no statistical variation was observed (P>0.05). And in lower-risk MDS the expression levels of HO-1protein, p-ERK and p-JNK increased and the expression levels of p-p38MAPK decreased gradually in a dose-dependant manner (P<0.05).3. In lower-risk MDS patients after treating BMMNCs with20μM zinc protoporphyrin-IX (ZnPP-IX) and20μM hemin, the expression levels of HO-1protein, p-ERK and p-JNK decreased and the expression levels of p-p38MAPK increased compared to the group of20μM hemin (P<0.05).Conclusions:1. The expression levels of HO-1protein is an independent factor which can be used to estimate the prognosis of lower-risk MDS. Upregulation of HO-1may participate in delaying the progression of lower-risk MDS. HO-1improve the prognosis of lower-risk MDS, which probably associated with its role in prolonging the duration of progression-free survival and promoting erythroid differentiation and hemoglobinization.2. HO-1as a cytoprotective factor contributes to inhibiting p38MAPK and activating ERK and JNK signaling pathway in lower-risk MDS. High expression of HO-1in lower-risk MDS was associated with good prognosis, and the effect of HO-1on the progress of MDS might be involved in MAPK signaling pathway. Upregulating the expression of HO-1may be a new targets to improve the prognosis of MDS by promoting effective hem.