Expression Of TFPI-2Gene And Its Promoter Methylation In Acute Myeloid Leukemia

BACKGROUND In recent years, so many studies showed that the DNA methylation especially the hypermethylation of tumor suppressor genes play an important role in the pathogenesis, progression, diagnosis and prognosis of leukemia. Tissue factor pathway inhibitor-2(TFPI-2) is a protease inhibitor with three Kunitz domains and belongs to the super family of serine protease inhibitor. It plays a major role in extracellular matrix (ECM) degradation during development, tumor cell invasion and metastasis, wound healing and angiogenesis. As a kind of tumor suppressor gene, TFPI-2can silence the gene expression when it gets hypeymethylation in many humun tumors such as the non-small cell lung cancer, hepatocellular carcinoma, prostate carcinoma, et al, leading to the loss of cytostatic and promopting cell apoptosis to the tumor. Now there are rare studies about the hypermethylation of the TFPI-2gene in acute myeloid leukemia. In this study, we investigated TFPI-2mRNA expression and promoter CpG island methylation status of patients with acute myeloid leukemia at onset, remission and relapsed/refractory stages, analyzed the relationship with clinical characteristics, and explored the clinical significance of TFPI-2in AML.Part I Expression of TFPI-2Gene in Acute Myeloid LeukemiaOBJECTIVE To detect the mRNA expression and clinical significance of TFPI-2in bone marrow mononuclear cells from patients with AMLMETHODS 1. Bone marrow mononuclear cells were isolated from newly diagnosed AML (n=33), complete remission AML (n=19), relapsed/refractory AML patients (n=12) and iron deficiency anemia patients (control group, n=15). Expression of TFPI-2mRNA was detected with real-time quantitative PCR (RT-PCR). The relationship between TFPI-2expression and AML patients’clinical characteristics has been analyzed.2. According to their response of chemotherapy, the newly diagnosed AML patients were divided into two groups:complete remission group (CR) and non-complete remission group (PR+NR), levels of TFPI-2mRNA expression between the two groups were compared by Mann-Whitney U Test.3. All the statistical analyses were carried out using SPSS17.0. The significance of different groups was evaluated by medians and P<0.05was considered statistically significant.RESULTS1. Our results showed that the expression of TFPI-2mRNA in newly diagnosed AML, complete remission AML and relapsed/refractory AML patients was significantly lower than that in the controls (P<0.05). Furthermore, its expression in relapsed/refractory AML patients was lower than that in newly diagnosed AML patients (P=0.006). Compared with complete remission AML patients, the expression of TFPI-2mRNA in newly diagnosed AML patients was significantly reduced (P=0.030).2. Observation of the relationship between TFPI-2expression and therapeutic efficacy in AML:After two courses of chemotherapy, the level of TFPI-2mRNA was much higher in the CR group than in the non-CR group (P=0.031). In23cases of complete remission (CR) AML patients, TFPI-2mRNA expression exhibited a trend toward elevation after CR compared with before chemotherapy treatment (P=0.044); In10cases of non-CR AML, TFPI-2mRNA expression levels after two standard chemotherapies were compared with before treatment, but no statistical significance was found (P>0.05).3. This study followed four patients who were newly diagnosed AML. For the first and the second patients, TFPI-2expressed higher and the patients were in the persistently CR statement after chemotherapeutics. The expression of TFPI-2was kept at a low level in the third patient, who died of ineffective treatment. The forth patient who was in the CR statement after chemotherapeutics, had the continuously high level of the expression of TFPI-2, and the expression of TFPI-2mRNA displayed remarkably low after receiving five months chemical treatment. Subsequently, this patient relapsed one month later.4. There was no correlation of TFPI-2gene mRNA expression level with gender, age, peripheral WBC (white blood cell) counts, FAB subtype and chromosome karyotypes in AML patients (P>0.05).CONCLUSIONS1. The expression of TFPI-2mRNA in newly diagnosed AML, complete remission AML and relapsed/refractory AML patients was much lower than that in controls (P<0.05), which showed that the reduced TFPI-2expression level was possibly related to the occurrence and prognosis of AML.2. The levels of TFPI-2mRNA were much higher in the CR group than in the non-CR group, suggesting that high expression levels of TFPI-2indicate a high CR rate. Besides, TFPI-2mRNA expression levels in patients with CR were significantly higher than that in patients before chemotherapy, which further illustrating that the expression of TFPI-2gene was associated with the occurrence and progression of AML.3. The low-level expression of TFPI-2of AML patients before chemotherapeutics may have a higher TFPI-2expression level after receiving chemotherapeutics and CR. For those who did not receive CR, the expression of TFPI-2was kept at a low level. What is more, for the AML patients who acquired CR, the expression of TFPI-2may decrease again before relapse. It is significant to observe the early relapse and judge prognosis of AML patients by detecting the expression level of TFPI-2.4. There was no correlation of TFPI-2gene expression level status with gender, age, peripheral WBC (white blood cell) counts, FAB subtype and chromosome karyotypes in AML patients (P>0.05), which demonstrate the methylation status of TFPI-2may be independent predictors of AML. Part II Detection and analysis of Aberrant Methylation of TFPI-2in Acute Myeloid LeukemiaOBJECTIVE This study aims to detect the methylation status in promoter region of tissue factor pathway inhibitor-2(TFPI-2) gene in bone marrow cells from acute myeloid leukemia(AML) patients and discuss its role in oncogenesis and progression.METHODS1. The methylation pattern in promoter region of TFPI-2gene was detected with Methylation-specific PCR (MSP) in64clinic bone marrow samples including33newly diagnosed AML patients,12relapsed/refractory patients,19complete remission patients.15iron deficiency anemia patients were assigned to the control group.2. All the statistical analyses were carried out using SPSS17.0. The significance of different groups was evaluated by medians and P<0.05was considered statistically significant.RESULTS1. The percentage of TFPI-2promoter methylation in AML patients was64.63%(42/64). In newly diagnosed AML group, complete remission AML group and relapsed/refractory AML group,the percentages of TFPI-2promoter methylation were66.67%(22/33),52.63%(10/19) and83.33%(10/12), respectively, and there was no statistical significance between them(P>0.05). None of the control patients mononuclear cells showed methylation of the TFPI-2gene.2. The optical density ratio of TFPI-2mRNA expression was0.165(0.005-2.099) in methylated AML patients, and0.597(0.011-2.787) in unmethylated AML patients, there was a significant difference between the two groups(P<0.05). 3. There was no correlation of TFPI-2gene promoter methylation status with gender, age, peripheral WBC (white blood cell) counts, hemoglobin, platelets, FAB subtype, chromosome karyotypes and diagnostic staging in AML patients (P>0.05).CONCLUSIONS1. The percentage of TFPI-2promoter methylation in AML patients was64.63%(42/64). TFPI-2methylation frequency in refractory AML group was higher than that in newly diagnosed AML group and complete remission AML group, but there was no statistical significance (P>0.05). None of the control patients mononuclear cells showed methylation of the TFPI-2gene. The data showed that abnormal methylation of TFPI-2gene promoter may be closely related to AML and participate in the progress process. Both high methylated ratio and low expression of TFPI-2can indicate poor prognosis, therefore, monitoring the index has great significance for the selection of chemotherapy regimens of AML.2. The optical density ratio of TFPI-2mRNA expression was lower in methylated AML patients than that in unmethylated AML patients(P<0.05), which demonstrate that the abnormal methylation of its promoter CpG island may be one of the main reasons that leading to the decrease even the silence of the TFPI-2gene expression.3. There was no correlation of TFPI-2gene promoter methylation status with gender, age, peripheral WBC (white blood cell) counts, hemoglobin, platelets, FAB subtype, chromosome karyotypes and diagnostic staging in AML patients (P>0.05), which demonstrate the methylation status of TFPI-2may be predictors independent of these clinical variables.