| ObjectiveThe pathogenesis and development of acute leukemia is a complex process,many intracellular signal transduction factors and gene transcription regulatory factors participate and play important roles in it.Beta-catenin is a kind of cytoskeletal proteins, which mediate intracellular adhesion.Meanwhile,it also participate in the regulation of multiple gene expressions as an important signal transmission factor in wnt signal pathway.The abnormal accumulation of beta-catenin may up-regulate its downstream target genes in wnt pathway,promote cellular malignant transformation and production of tumor.As a downstream target gene of Wnt/β-catenin pathway,CyclinD1 promote cell transformation from G1 phase to S phase.The abnormal expression of cyclinD1 will cause cell cycle out of control,which produces abnormal cellular proliferation and tumor genesis.Aberrant activation of Wnt/beta-catenin signaling is closely associated with pathogenesis of many solid tumors,such as rectal or colon cancer,breast cancer et al.However,the related study in acute myloid leukemia is not penetrating.This study applied method of real-time fluorescence quantitative RT-PCR to detect the level of beta-catenin and cyclinD1 mRNA in various subgroups in acute myeloid leukemia,and analyse the potential significance.There was no related literatures reported in the world,this study will provide new theoretical basis in revealing the pathogenesis of AML.Methods1.patient selectionCollect EDTA anticoagulated bone marrow samples 3-5ml from clinic and hospitalized newly diagnosed acute myeloid leukemia patients in Blood Disease Therapeutics Center of China Medical University affiliated Shengjing Hospital during September 2006 and May 2008,among which there was 5 cases of AML-M1,15 cases AML-M2,11 cases of AML-M3,13 cases of AML-M4,8 cases of AML-M5,3 cases of AML-M6,55 cases totally as experimental group.Collect EDTA anticoagulated bone marrow samples 3-5ml from 12 cases of Benign blood disease patients as control group.2.experimental methodMononuclearcell(MNC) was separated from anticoagulated bone marrow samples by Ficoll-Hypaque density gradient centrifugattion;extraction of total RNA;reverse transcription reaction system was disposed as 20μl to synthesize cDNA;primer was designed and synthesized by Takara company,SYBR Green I fluorochrome real time quantitative RT-PCR method was apllied to detect expression level of beta-catenin and cylinD1,the concrete procedure follows reagent instructions,GAPDH was detected as inner reference.3.Result determination8 fold attenuate the cDNA of standard samples in 5-6 concentration gradient,take 2μl as template to run real time PCR reaction respectively.Standard curve was draw by each CT value of amplification curves.Samples copy number was obtained according to standard curve.GAPDH was detected as inner reference,while beta-catenin and cyclinD1 were detected as objective genes.Relative quantitative comparisons among different samples were taken according to the copy number ratio of objective gene and inner reference gene.4.Statistical methodsAll the data were analyzed with SPSS for windows 13.0 software.Relative expressive quantity of objective gene was taken as reference values,Mann-Whitney test and Kruskal-Wallis test in nonparametric statistics was applied in the comparisons among various groups,P value of less than 0.05 was accepted as statistically significant. Bivariate linear was used to analyze the correlation analysis.Results1.Reliability and accuration of method(1)purity and quality analysis of RNA Detecting extracted total RNA by ultraviolet spectrophotometer,the ratios were between 1.8 and 2.0.further identification was done by agarose gel electrophoresis, bands were clearly,which shows no obviously degradation(2)specificity of amplification and accuration of quantitationStandard samples undertook PCR reactions after gradient dilution,which was aimed to make standard curves.All the correlation coefficients were more than 0.998,slopes were between-3.3 and-3.5 of standard curves,indicating quantitative accuration and good amplified effects.Dissociation curves show the identical Tm value for all samples,and the sharp sole peak ensured the specific amplification of target sequence without primer dimer.2.Expression of beta-catenin gene in various subtypes of acute myeloid leukemiaBeta-catenin mRNA expression levels in 55 cases of AML mononuclearcell was statistically higher than that in 12 cases of benign blood disease patients (p=0.004),among which AML-M4,M5 shows dramaticly high expression(median was 1.72×10-2and 1.735×10-2 respectively),while AML-M3 shows low expression(median was 0.303×10-2).There was no statistical difference between AML-M3 and other various subgroups ofAML(p＞0.05).3.Expression of cyclinD1 gene in various subtypes of acute myeloid leukemiaCyclinD1 shows overexpression in various subtypes of AML in different extent,which were dramaticly higher than that in benign blood disease group.Among which AML-M2 shows high expression(median was 4.82×10-4),AML-M3 shows low expression.There was statistical difference between AML-M3 group and AML-M6 group(p=0.038),but no statistical difference among other subtypes of AML (p＞0.05).There were low level expressions of cyclinD 1 mRNA in 12 cases of benign blood disease patients as control group.4.Correlation analyses of beta-catenin and cyclinD1 gene expressions in various subtypes of AMLWe analyze correlation between beta-catenin and cyclinD1 expressions in 55 cases of acute myeloid leukemia in various subtypes(AML-M15cases,AML-M215 cases, M311 cases,M413 cases,M58 cases,M63 cases).resutts shows the expression level of beta-catenin and cyclinD 1 were correlated with each other in AML-M1,M2 and M4(r value is 0.822,0.627,0.712 respectively;p value is 0.001,0.020,0.002 respectively),while there were no obviously correlationship of beta-catenin and cyclinD1 in AML-M3,M5,M6.ConclusionThe patients of AML have prominent overexpression of beta-catenin and cyclinD1,which has significant correlationship in part of the subgroups.It is considered that Wnt/beta-catenin pathway is aberrantly activated in AML,which probably active the downstream target gene cyclinD1 to participate in the regulation of confused cycle and abnormal proliferation of leukemic cells.
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