The Study On The Expression Of Ang2 Gene And Its Clinical Significance In Acute Myeloid Leukemia Cells

Background and objective:Acute myeloid leukemia(AML) is a commen malignant tumor that has seriously harmed human healthy. The current standard chemotherapy regimen can gain 60%-80%of complete remission (CR), even as part of patients are difficult to achieve CR and become refractory leukemia, eventually death in invalid treatment. Correct prognostic appraisal of AML plays an important role in formulating perfect therapeutic strategies, performing individual treatment, improving the therapeutic efficacy. In the past, elder, high white blood cell(WBC) count, long duration to complete remission, secondary leukemia and high-risk cytogenetic characteristics were considered to be affirmative factors to poor prognosis of AML, and karyotype was considered to be the most important and independent prognostic factor. With the deepening of study on the molecular mechanism of leukemia and development of molecular biologic technique, many gene mutations and abnormal gene expressions were found to be related with prognosis of AML, which replenished the prognostic appraisal of AML and provided valuable indexes especially for AML patients with normal karyotype or intermediate-risk karyotype. For example, AML with CEBPA mutation or NPM1 mutation had a better prognosis, while AML with P53 mutation, MLL mutation or FLT3 mutation had a worse prognosis. And overexpression of Sorcin, EGR, BAALC or MN1 were worse predictors for AML with normal karyotype.According to the reports and bioinformatic analysis, Ang2 gene localized on 8p21 and contained 8 introns and 9 exons,496amino acids,relative molecular weight of about 75.000,about 60%is same with angiopoietinl(Angl),and also similar to the molecular structure of Angl. Ang2 is an endothelial-sppecific groeth factor,mainly expressed in active site and a high degree of proliferation of tumor blood vessels, may be early signs of tumor angiogensis,its expression rapidly increased the tumor growth and prone to early signs of a shift. Ang2 gene is high expression in gastric cancer, breast cancer, liver cancer, hemangiomao or gliomas, and closely related with the progress of gastric cancer. Ang2 is high expression in the CD7 (+) AML, while low expression in the t (8; 21) AML, analysised by reverse transcriptase PCR(RT-PCR). Ang-2 expression level is reduced when achieved complete remission after chemotherapy and not easy to detect. Immunohistochemical analysis confirmed that Ang2 is high expression in bone marrow of AML patients, and is higher expression in actue phase than chronic phase of chronic myeloid leukemia (CML). Martinelli et al determination Ang2 expression of early B-cell chronic lymphocytic leukemia (CLL), results showed that high expression of Ang2 related with IgV (H) gene mutation, bone marrow angiogenesis in early B-cell chronic lymphocytic leukemia.So, respecting the expression and function of Ang2 gene in solid tumors, we explored Ang2 gene expression level in AML by RQ-PCR, in order to further confirm the inflounces of Ang2 to the early diagnosis and prognosis of AML,and whether the expression level in various types showes significant difference.METHODS1. Study subjects:All AML bone marrow specimens were taken from 139 cases of diagnosed patients with the clinical manifestations, cell morphology and immunophenotype and cytogenetic features. Control group are age, sex matched 16 healthytransplantation donors.2. Primer design and synthesis:In accordance with the GeneBank sequence we design Ang2 gene and the housekeeping genesβ-actin primers.3. Extraction of bone marrow mononuclear cells from newly diagnosed AML and non-hematologic malignancies patients, then extraction of total RNA, reverse transcription into cDNA, doing real-time fluorescence quantitative PCR finally. To determine the specificity of PCR product through the melting curve. Take an average of two Ct values 2-ACT value, as the specimen relative mRNA expression levels,ΔCT= (target gene CT value-reference gene CT value). Comparison the gene expression differences of patients bone marrow mononuclear cells from untreated AML group and the normal control group, untreated group and remission group, relapse group and the normal control group, relapse group and the remission group, refractory group and the normal control group, refractory group and remission group, untreated group and the relapse group, untreated group and the refractory group, various subtypes and normal control groups. And relative Ang2 mRNA expression levels are divided into high expression group and low expression groups. Comparison the number of cases from each treatment group in the first CR cases and NR cases, refractory cases and non-refractory cases, died within 3years and cases 3 year still alive.4. All statistical analyses were performed with the SPSS 13(SPSS Inc, Chicago, IL, USA). Mann-Whitney U test was used to analyze the difference in the expression of each gene between each group. Rate compared using x2 test Correlations between variables were assessed by the Spearman rank correlation. Kaplan-Meier estimation was used to plot survival curves, and log-rank tests were used to test the difference between groups. Two-sided P values<0.05 were considered statistically significant.Results:1. Analysis of purity and quality of RNA and cDNA:total RNA and cDNA were detected by UV Spectrophotometer, and the ratio are between 1.8 and 2.0.2. The same cDNA sample was diluted by 2-fold, was setted by 6 gradients. The concentration gradients were measured, as the scope is 8.1～258.8ng/μl. The housekeeper gene and target genes were amplified respectively. The result is that 6 gradient ACT differences are very small, thus amplification efficiency of the housekeeper gene and target genes are identical at the concentration range. Melting curve was done after amplification. It showed sharp single peak and no other specific peak with each gene. So we considered PCR products were homogeneous,no other non-specific amplification and primer dimmer. We can see that a single strip, no primer dimer and non-specific amplification by agarose gel electrophoresis.3. Gene expression:Ang2 expression level in untreated AML group was significantly higher than the normal control group,remission group (Z=-2.570, P=0,010; Z=-3.470,0.001),and in relapse group was significantly higher than untreated group and normal group (Z=-2.142, P=0.013; Z=-2.942,0.003); in refractory group was significantly higher than untreated group and normal group (Z=-3.549, P=0.000; Z=-3.322,0.001); Between untreated AML group and relapse group,refractory group showed no significant difference(P>0.05). Among various subtypes,various subtypes and remission group,various subtypes and normal control group showed no significantly difference(P>0.05) except the Ang2 gene expression level was higher in M2 than remission group.4. Relationship between gene expression level and clinical indicators: Correlation analysis was done between expression level of Ang2 gene and clinical indicators such as age, hemoglobin, white blood cells, platelets, lactate dehydrogenase level and the proportion of leukemic cells in peripheral blood and bone marrow leukemic cells of newly diagnosed patients. The results showed that age, hemoglobin, white blood cells, platelets, lactate dehydrogenase level and bone marrow leukemic cells were not significantly related to Ang2 expression level, the absolute value of correlation coefficients were less than 0.3, P>0.05. Ang2 expression level between man and woman had no significantly difference (Z=-0.587,P=0.557)5. Relationship between gene expression level and efficacy:The efficacy of M3 patients was superior to other subtypes because of molecular targeted therapy, so M3 cases are excluded in efficacy analysis. According to the median of each gene expression level, patients are divided into two groups. Removed the lost cases, cases which can be analyzed treatment response Ang2 are 62 cases of high expression group,49 cases of low expression group. There are 32 cases (51.6%),30 cases (71.4%) in high-expression group and low-expression group achieved CR,that showed significant diference(P<0.05). There are 9 cases of refractory cases (14.5%), 1 cases (2.1%) in high-expression group and low-expression group. There was significant difference between refractory cases of high-expression group and refractory cases of low expression group (P< 0.05).79 cases have been followed-up more than 3 year,54 cases of high expression group,28 cases died within3 year (51.6%); 25 cases of low expression group,7 cases died within 3 year (29.4%). There was signifinant diference between high expression group and low expression group (P<0.05).Conclusion:1. The extracted RNA, cDNA are of high purity, good quality and can be used for follow-up experiment2. Designed primer of each gene is specific. And its amplification efficiency is consistent with housekeeper gene. 3. Ang2 mRNA expression level are significantly higher in untreated AML group,relapse group,refractory group than the normal control group,remission group.Among various subtypes,various subtypes and remission group,various subtypes and normal control group showed no significantly difference except the Ang2 gene expression level was higher in M2 than remission group.4. Correlation analysis showed that age, hemoglobin, white blood cells, platelets, lactate dehydrogenase level and bone marrow leukemic cells were not significantly related to Ang2 expression level. Ang2 and the proportion of leukemic cells in peripheral blood was negative correlated.5. Ang2 gene may be assessed as an independent indicator of early diagnosis.6. Ang2 gene expression levels may serve as indicators for assessing response to treatment.7. Ang2 gene expression levels is related to survival time.