Research On The Expression Of UBAP1 Gene And Tumor-suppressor Gene P16 In Acute Leukemia

ObjectiveThe mechanisms of acute leukemia is not clear. It is well-established that abnormal cell cycle regulation, abnormal proliferation, differentiation barriers, blocked apoptosis and so all influence the development of acute leukemia. Numbers of factors are linked with it, i.e. cell cycle protein and related inhibitors, transcription factors and related inhibitors.UBAP1 is a new member in ubiquitin-related protein family. the structure of UBAP1 include multiple phosphorylation and glycosylation sites and UBA domain. UBA domain can mediated target protein and ubiquitin-protein non-covalent binding which ubiquitins target protein. And then ubiquitined target protein is degraded by the 26S proteasome. p16 is a member of the cyclin dependent kinase inhibitor family. It can inhibite cell proliferation by effectively preventing the cells from G1 phase to S phase.UBAP1 gene and p16 gene are located on chromosome 9 with p21-22. The expression of UBAP1 gene in malignant tumors are different. But the expression in acute leukemia has not been studed. The expression p16 gene in many malignant tissue is lower and it is a tumor suppressor gene. The lower expression effects the incidence of acute leukemia. In the paper, we studied the expression of the UBAP1 gene and p16 gene in acute leukemia and the correlation between the UBAP1 and p16. Study of this relationship will help us to understand the underlying mechanisms of acute leukemia and eventually contribute to the new stratages of prevention and therapy of acute leukemia.Methods1. Collecting EDTA anticoagulated bone marrow samples 3-5 ml, which are from clinic and hospitalized newly diagnosed acute leukemia patients in Blood Disease Therapeutics Center of China Medical University affiliated Shengjing Hospital during August 2008 and August 2009.56 cases are totally as experimental group and 22 cases of Non-hematologic malignancies are as control group. Using Ficoll lymphocyte separation medium extractes Mononuclear cells(MNC) from bone marrow samples, then saline washes twice. Each (2-8)×106 cells are added 1ml Trizol reagent, frozen into the-80℃refrigerator.2. Using Trizol, Chloroform, Isopropyl alcohol and 75% ethanol extractes total RNA of samples, and using protein and nucleic acid analysis of the DU800 detectes RNA purity, purity selected between 1.8 and 2.0 RNA. In accordance with the TaKaRa RT kit instructions, using reversed transcription (RT) synthesizes cDNA, frozen into the-80℃refrigerator.3. The gene of UBAP1 and p16 are as purpose gene, and GAPDH used as a housekeeping gene. Primers were designed and synthesised by the Takara Biotechnology Co Ltd. quantitative reaction kit was provided by the TaKaRa. using fluorescent dye SYBR Green I real-time quantitative PCR method determined the expression of UBAP1 and p16. The concentration of standard was diluted from 4 to 5. 2μl of each concentration taken as templates were amplified. Then building the standard curve of the target gene and housekeeping gene through quantitative analysis software. according to the standard curve to read the sample copies, and eventually the ratio of the target gene and housekeeping gene copies were as the relative amounts of mRNA samples. The PCR products were separated by 1.5% agarose gel electrophoresis, and captured by ultraviolet light camera observation.4. Collecting heparin anticoagulated bone marrow samples 2-3 ml over the same period patient group. preparation the chromosomes by using the direct method and 24h cultured, droping piece, R-banding, Giemsa staining, and Olympus BX51 microscope karyotype analysis. According to "international human cytogenetics naming system (ISCN 2005) "described karyotype.5. SPSS16.0 statistical software used for data analysis, p<0.05 as statistically significant. Kolmogorov-Smirnov Z rank test (z statistic with that) was used to compare mRNA expression levels of UBAP1 gene and p16 gene, Kruskal-Wallis H rank sum test was used to compare the expression of two genes between the chromosome prognosis groups. Spearman correlation test was for the mRNA expression level of correlation of UBAP1 gene and p16 gene.Results1. Newly diagnosed AL were collected 58 cases, Non-hematologic malignancies was 25 cases. Total RNA of mononuclear cells were extracted. DU800 nucleic acid protein analyzer determined the amount of RNA. The purity of the RNA between 1.8 and 2.0 was for reverse transcription. And eventually collecting cDNA samples were 78 cases, of which 56 cases were patient group,22 cases were the control group.2. Successfully establishing a standard curve (R2= 0.9996, slope:-3.29). According to the standard curve reading the target gene and housekeeping gene copies. Through the ratio of the target gene and housekeeping gene copies obtaining the relative amounts of mRNA. The melting courve of target gene showed a single peak, indicating PCR products were homogeneous, no nonspecific amplification and primer dimer. The PCR products were separated by 1.5% agarose gel electrophoresis, and captured by ultraviolet light camera observation. The result further verified the fragment length of PCR products consistent with the experimental design.3. Totally preparation of chromosome samples were 41 cases,38 cases of which can be used for karyotype analysis. According to SWOG criteria for prognosis group.4. Statistical analysis:Compared with the control group, the UBAP1 gene in acute leukemia patients is highly expressed (p<0.01), while the p16 gene is lowly expressed(p<0.01). We have divided these cases into groups with FAB method and each group is compared with the control group. We found that all groups show significant difference (p<0.05) except M1, M2 and M3 group in p16 gene; For UBAP1 gene, After typing with FAB method, only M4 and M5 have significant difference in adult group(p<0.05). The expression of UBAP1 gene and p16 gene mRNA in SWOG chromosome prognosis groups was not significant difference (p>0.05). A negative correlation (r=-0.827, p<0.01) is found on the mRNA expression level of UBAP1 gene and p16 gene in the control group, but there is not the negative correlation in patient group(r=0.038,p>0.05).ConclusionsThe upregulation of UBAP1 gene and the downregulation of p16 gene may same participate in the pathogenesis of acute leukemia. High expression of UBAP1 gene major impact of the M4 and M5 subtypes in AML. This discovery will further explore the pathogenesis of acute leukemia and provide important theoretical basis for the target phase treatment. The abnormal expression of UBAP1 gene and p16 gene in acute leukemia is not associated with SWOG chromosome prognosis groups, explaining the expression of two genes are not effected by genetic changes.