| ObjectiveTo investigate the expression of homeobox A9(HOXA9) in myelodysplastic syndromes(MDS) patients and its specific pattern at both protein and RNA levels which might be associated with clinical characteristics and treatment response. In this study, protein level of HOXA9 was detected using flow cytometry in bone marrow samples. The expression of HOXA9 gene and how it altered with clinical features was measured by real-time quantitative PCR(RQ-PCR). Collectively, the specific effect of HOXA9 in MDS patients and its potential clinical values will be explored in this study. MethodsThe study group was comprised of 33 MDS patients. The positive control subgroup was comprised of 12 patients with acute myeloid leukemia(AML). The negative control ones were 20 patients with immune thrombocytopenia(ITP) and 18 normal controls. FACSCalibur flow cytometric instrument was used to detect the percentage of HOXA9+ cells in CD34+ cells as well as CD34+CD38- cells. We measured HOXA9 protein levels in nucleus and analyzed them based on WHO category, IPSS score and decitabine-treatment response in order to find the prognostic and therapeutic benefits of HOXA9. RQ-PCR with SYBR Green-based assays was used to detect the relative expression level of HOXA9 mRNA. We analyzed the expression of HOXA9 gene based on WHO category, IPSS score and decitabine-treatment response to find the potential prognostic and therapeutic benefits of HOXA9. ResultsFlow cytometry assays indicated that HOXA9 were highly expressed in both MDS and AML. The percentage of HOXA9+ cells in CD34+ cells in MDS cases was(37.88±27.74)%, which was significantly higher than that(20.31±20.49)% in control group(P<0.05). The ratio of HOXA9+/CD34+ did not show significantly difference between ITP and control groups(15.94±12.80 vs 20.31±20.49,P=0.78) as well as MDS and AML groups(37.88±27.74 vs 42.60±31.63,P=0.75). In CD34+CD38-progenitor pool, the ratio of HOXA9+/CD34+CD38- closely mirrored HOXA9+/CD34+ ratio, further conforming the overexpression of HOXA9 in malignant hematological diseases. The ratios of HOXA9+/CD34+ and HOXA9+/CD34+CD38- before decitabine treatment were(50.64±27.59)% and(55.67±28.57)%, which were both significantly higher than those(20.31±20.49 for HOXA9+/CD34+ and 20.16±18.79 for HOXA9+/CD34+CD38-) in normal control group(P<0.05). After decitabine treatment, we found statistically significant lower expression of HOXA9 than pre-treatment(22.57±19.35 for HOXA9+/CD34+, 22.42±20.74 for HOXA9+/CD34+CD38-, P<0.05) and both HOXA9+/CD34+ and HOXA9+/CD34+CD38- levels confirmed this trend.The relative expression level of HOXA9 mRNA in MDS was higher than normal, while there was no significant difference yet. Based on morphological category, the expression of HOXA9 revealed significant differences among refractory cytopenia with unilineage dysplasia(RCUD), refractory anemia with ringed sideroblasts(RARS), refractory cytopenia with multilineage dysplasia(RCMD), refractory anemia with excess blasts-1(RAEB-1) and RAEB-2(P<0.05). The expression of HOXA9 differ as the subtype of MDS changed, especially among RCMD to RAEB-2(3.91±2.84 vs 66.48±66.68,P<0.05). Consistent with the expression of HOXA9 protein, the expression of HOXA9 before decitabine treatment was significantly higher than that in normal controls(55.80±67.88 vs 5.50±4.47, P<0.05), while after decitabine treatment the expression of HOXA9 became lower(10.32±12.42). The m RNA level of HOXA9 was positively correlated with the proportion of bone marrow blast(r =0.70, P<0.05). ConclusionWe found that HOXA9 in MDS patients had an overexpressed pattern on both the protein and mRNA level. In addition, HOXA9 was positively correlated with the proportion of bone marrow blast and differed by WHO category of MDS as well as IPSS score. HOXA9 may work as a biomarker which shows malignant probability in patients. Interestingly, HOXA9 in pre-treatment cases was high, while decreased after decitabine-treatment, the karyotype and IPSS score improved as well. |
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