Expression Of Homeobox A6 In The Proliferation Or Differentiation Of Cells HL60 And Treatment Research

bjective:This topic mainly discusses expression on homeobox gene (HOX) HOXA6 and its protein in the proliferation or differentiation of HL60,and discusses its expression in following the intervention of use all-trans retinoic acid (ATRA) and/or arsenic trioxide(ATO),and explore HOX gene mediated pathogenesis of leukemia in gene level.Methods:1.HL60 cell line was purchased from Wellcome Bio Co.,Ltd..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, the final concentration of 1×10-6mol/1. (3)ATO group:Add the diluent of ATO, the final concentration of 1×10-6mol/1. (4) ATRA+ As2O3 group:while adding ATRA and ATO, the final concentration of 1×10-6mol/1.3.By the tumor cells culture techniques in vitro,and HL-60 cells continued to interfere with ATRA, and (or)ATO,then the formation conditions of normal group, ATRA group,ATO group, ATRA+ATO group of HL60 cells in culture were observed on the first,second,and third day.4.Their growth and differentiation was determined by Wright Giemsa staining.5.All group were extracted from total cellular RNA on 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.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 HOXA6.7.Randomly selected reverse transcriptase of the HOXA6 gene electrophoresis respectively HOXA6 gene in 1 day,2 days,3 days electrophoresis.The HOXA6 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.8.Statistical Methods of PCR:Results relative DNA copy number and RNA expression of the relative ACTB gene amount of (2-△Ct) said that the relative expression of HOXA6 gene, using the mean plus or minus standard deviation(X±S) that HOXA6 gene changes.Homogeneity of variance test, REPEATED MEASURE analysis of variance, Completed by the statistical software SPSS 17.0.9.Each group were lysed extract total cellular protein on the first day, the second day and the third day.10.Western blot:cells were detected in the HOXA6 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.11,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.12.statistical methods of Western-blot:analysis of cells in each group HOXA6 protein expression differences, all data with x±s, using REPEATED MEASURE analysis of variance, P<0.05 was statistically significant, P<0.01 for the significant.Results:1.Cell morphology identification, Wright's Giemsa staining demonstrated that the cultured cells are granulocytes.2.1% formaldehyde denaturing agarose gel electrophoresis showed that RNA electrophoresis of 5s, 18s,28s 3 band type and tidy, no significant tailing and dispersion, indicating structural integrity of RNA, no obvious degradation.3.RT-PCR amplification of the three groups were within the target gene HOXA6 and reference ACTB cDNA gene product, compared with the DNA molecule Marker said the size of PCR products were:HOXA6 to 126 base pairs (bp), ACTB gene 111bp, consistent with the intended size of the theoretical value.4.Different concentrations of ATRA-treated cells after 24h,in addition to 5×10-6mol/l outside the other concentrations are significantly inhibit cell proliferation. After the cells were treated with different concentrations of ATO, in addition to 10-7mol/l outside the other concentrations are significantly inhibited cell proliferation,and the greater the concentration of inhibition occurs earlier, the effect became. Different concentrations of ATRA,ATO combined treatment cells were markedly inhibited cell proliferation,and the greater the concentration of inhibition occurs earlier, the effect became.5.In normal group,the expression of HOXA6 gene and HOXA6 protein is more and more stronger when the time goes by.6.In ATRA group, the expression of HOXA6 gene and protein is always stronger than normal group in all three days, on the second day, the expression is the strongest.7.In ATO group and ATRA+ ATO group,the expression of HOXA6 gene and HOXA6 protein were were upregulated dramatically on the first day(higher than normal group) and downregulated on the secondly two days(lower than normal group). 8.Compared with normal group, HOXA6 gene and HOXA6 protein were upregulated by ATRA,and downregulated by ATO and ATO+ATRA.Conclusion:1.1 X 10-6mol/l ATRA, ATO and ATRA+ATO can promote HL-60 cells to mature.2. HOXA6 gene and HOXA6 protein were expressed in human myeloid leukemia cells HL60 and upregulated when the cells' proliferation.which means HOXA6 can enhance the proliferation of cell and this gene may be cause leukemia.3 ATRA can upregulate the expression of HOXA6 gene and protein in the three observed days,so the mechanisms of treatment of leukemia by ATRA may be associated with the regulation of the expression of HOX genes.4.In the differentiation of HL60,ATO downregulated the expression of HOXA6 gene and protein,so the mechanisms of treatment of leukemia by ATO may be associated with the downregulation of the expression of HOXA6 genes.