Expression Of Homeobox A9 In Myeloid Leukemia Cells HL-60

bjective:This topic mainly discusses expression on homeobox gene (HOX) HOXA9 mRNA and it's protein in myeloid leukemia HL-60, and discusses its expression in following the intervention of use all-trans retinoic acid (ATRA) and/or arsenic trioxide (As2O3), and explore HOX gene mediated pathogenesis of leukemia in gene level and protein level. Methods:1. HL-60 cell line used as a model of leukemia.2. Experimental groups. Were divided into 4 groups:(1) control group (Normal):no drug, on behalf of the same amount by adding RPMI 1640 medium basic training system. (2) ATRA group:Add the diluent of ATRA. (3) As2O3 group:Add the diluent of As2O3. (4) ATRA+As2O3 group:while adding ATRA and As2O3.3. Determine the final concentration of drug by Experiment on cell proliferation and inhibition.4.By the tumor cells culture techniques in vitro, and HL-60 cells continued to interfere with ATRA, and (or) As2O3,then the formation conditions of normal group, ATRA group, As2O3 group, ATRA+As2O3 group of HL-60 cells in culture were observed in the first, second,and third day.5. Their growth and differentiation was determined by Wright Giemsa staining.6. Observing the expression of HOXA9 mRNA by PCR.6.1 All group were extracted from total cellular RNA in the first, second, and third day, and total RNA was electrophoresed in 1% formaldehyde denaturalized agarose gel in order to validate integrity of RNA.6.2 By random primer to total RNA reverse transcriptase to cDNA, real-time fluorescent quantitative reverse transcription polymerase chain reaction (fluorogenic quantitive reverse transcription polymerize chain reaction, FQ-RT-PCR) to detect cell proliferation and differentiation process of gene expression in each group HOXA9.6.3 Randomly selected reverse transcriptase of the HOXA9 gene electrophoresis respectively HOXA9 gene in the first day, the second days, the third days electrophoresis. The HOXA9 gene cDNA and the ACTB gene positive product of the respective cDNA dilution standard curve production, according to the exponential growth cycle of each sample is the starting point of the cycle threshold (cycle threshold, Ct) and standard curve slope of the calculated volume of each sample of the starting cDNA template multiple values to ACTB gene were standardized as internal reference.6.4 Statistical Methods:Results relative DNA copy number and RNA expression of the relative amount of (2-ΔΔCt) said that the relative expression of HOXA9 gene, using the mean plus or minus standard deviation (X±S) that HOXA9 gene changes. Homogeneity of variance test, TOW-WAY AONVA analysis of variance, groups were compared with the number 22 LSD method, completed by the statistical software SPSS 17.0.7. Observing the expression of HOXA9 protein by Western blot.7.1 Each group was lysed extract total cellular protein in the first day, the second day and the third day.7.2 Western blot:cells were detected in the HOXA9 protein:determination of samples with the BCA kit total protein concentration of polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie brilliant blue staining observed protein bands and electricity transfer (electrotransfer), closed PVDF membrane, immune response (by adding an anti and two anti-), chemiluminescence showed that the target band.7.3 Image Analysis:Western blot results will be sent to the computer and use Quantity one image analysis software to analyze the protein bands, get integral optical density, after correction with the internal reference, the ratio of integral optical density to represent the relative protein expression.7.4 Western blot statistical methods: analysis of cells in each group HOXA9 protein expression differences, all data with x±s, using TOW-WAY AONVA analysis, P<0.05 was statistically significant. Completed by the statistical software SPSS 17.0. Results:1. Different concentrations of ATRA, As2O3 and ATRA+As2O3 treated cells, cell proliferation was inhibited in the various drug group of 10-5mol/l, cells did not increase and all died soon; the number of cells gradually increased in the various drug group of 10-6mol/l and 10-7mol/l, After cell growth to flatten the peak, The cell growth trend of Two concentrations was similar, Select 10-6mol/l as the final concentration of drug intervention by combining Literature at home and abroad and facilitate the experiment Katori reagent.2. Cell morphology identification, Wright's Giemsa staining demonstrated that the cultured cells are granulocytes. Normal cells had no obvious change in the comparison between in the first day and the third day; comparing in the first day and the third day, the cell of other groups were out of shape in the third day, the nucleus becomes irregular sizes, showing that nuclear lobulation, kidney-shaped nucleus, beans nuclear, nuclear was relatively narrow, the nuclear/cytoplasmic ratio became smaller, indicating the direction of cell differentiation to mature.3. In this study, the HOXA9 gene in each group have shown a tendency, first increased and then decreased, it has been expressed in the first day, expression has increased in the second day, while expression was decreased in the third day. The HOXA9 protein in each group has been expressed in the first day, expression of HOXA9 protein in Control group and ATRA group was first increased and then decreased, its were higher in the second day than in the first and third day, the expression of HOXA9 protein in As2O3 group and ATRA+As2O3 have shown a decreased tendency gradually.4. In HL-60 cells cultured in vitro process, continued intervention by adding 10-6mol/l ATRA,10-6mol/l As2O3,10-6mol/l ATRA+As2O3, the expression of HOXA9 genes in ATRA groups at different time points were higher than the normal group and the difference was statistically significant. The differences of HOXA9 gene expression between As2O3 and ATRA+As2O3 groups with the normal group was not significant in the first and third days, the expression of HOXA9 genes in As2O3 and ATRA+As2O3 groups were higher than the normal group in the second day and the difference was statistically significant. The expression of HOXA9 protein in ATRA groups at different time points were higher than the normal group and the difference was statistically significant, the expression of HOXA9 protein in As2O3 and ATRA+As2O3 groups were higher than the normal group in the first day and the difference was statistically significant, the expression of HOXA9 protein in As2O3 and ATRA+As2O3 groups were lower than the normal group in the second day and the difference was statistically significant, the differences of HOXA9 protein expression between As2O3 groups with the normal group was not significant in the third day, the expression of HOXA9 protein in ATRA+As2O3 groups were lower than the normal group in the third day and the difference was statistically significant. Conclusion:1.10-6mol/l ATRA,10-6mol/l As2O3 and 10-6mol/l ATRA+As2O3 can promote HL-60 cells to mature.2. HOXA9 gene and HOXA9 protein have expression in human myeloid leukemia cells HL-60.3. 10-6mol/l of ATRA could Up-regulate the expression of HOXA9 gene or HOXA9 protein in human myeloid leukemia cells HL-60,10-6mol/l of As2O3 and ATRA+As2O3 could regulate the expression of HOXA9 gene or HOXA9 protein in human myeloid leukemia cells HL-60, but no obvious regularity.4. HOXA9 gene may be one of the major regulatory genes in the process of human myeloid leukemia cells HL-60 cells to mature.5. The mechanisms of treatment of leukemia by ATRA, As2O3 may be associated with the regulation of the expression of HOX genes or HOXA9 protein.