| Background and ObjectiveHomeobox gene plays a key role in controlling biological development and cell differentiation,existing widely in living organisms.CDX2,caudal type Homeobox transcription factor 2,is an important gene regulating the development and growth from the mouth to the anus along longitudinal axis.First isolated from success in flies by Mlodzik,the CDX2 gene has 22～23kb,which locates in the chromosome 13 q12-13.It consists of three extrons and two introns,coding a protein with 311 amino acids.The protein bind to DNA in a manner of spiral-ring-spiral to regulate the expression of DNA,which play as a transcriptional factor.CDX2 is expressed in intestinal epithelium,pancreas,but not in breast,prostate,kidney,liver,bone marrow and peripheral.The abnormal expression of CDX2 may lead tumor development, together with other genes.Abnormal expression of CDX2 can be used to predicte malignant degree of tumors,and involved in infiltration,metastasis and prognosis. CDX2 is upregulated in the majority of cancers such as stomach cancer,esophageal cancer,gallbladder cancer and is expressed in low levels in colorectal cancer and colon cancer,detected by gene array,immunohistochemical and RQ-PCR.CDX2 is expressed in hematopoietic progenitor cells,and involves in cell differentiation and proliferation.Leukaemia will develop if CDX2 overexpressed.Research results show that abnormal expression of CDX2 in hematopoietic cells induced abnormal expression in the HOX family protein in mice.The abnormal expression of HOX leads tumor development including leukemia.Abnormal expression of Hox gene is detected in breast cancer,colon cancer,kidney,glioma,leukemia and tumor osteogenic sarcoma etc.Furthermore,CDX2 is expressed in 90%of patients with acute myeloid leukemia(AML),40%of patients with MDS and 20%of patients with CML.Growth inhibition and reduced clonogenicity of AML cell lines were observed following knockout of CDX2.A recent research shows that CDX2 is expressed in high level in ALL and CLL,related with poor prognosis.There are no similar reports in Chinese.In order to investigate the expression of CDX2 in leukemia,mRNA expression of CDX2 in bone marrow cells from patients with acute leukemia and chronic leukaemia patient,as well as chronic myeloid leukemia cell line-K562 cell will be assessed by RT-PCR.The relationship between the expression level of CDX2 and leukemia cell percentage in patients' bone marrow is analyzed.The possible role of CDX2 in the development and growth of leukemia will be discussed.Methods1.Research object1.1 Acute leukemia patients:Total 68 adult patients with acute leukemia were enrolled in this study,including 44 cases of de novo acute leukemia(32 casese of AML and 12 cases of ALL),10 cases of relapsed(5 casese of AML and 5 cases of ALL) and 14 cases of complete remission(9 casese of AML and 5 cases of ALL).All the patients,including 36 males and 32 females,are aged from 14-78 years old(mean 37 years old) and diagnosed according to the criteria for diagnosis and curative effects of hematological disease(3rd edition) by Zhang Zhinan.1.2 Chronic myelogenous leukemia:Total 21 cases of patients with chronic myelogenous leukemia were enrolled in this study,including 13 cases of chronic phase,4 cases of accelerated phase and 4 cases of blast phase.The patients had a median age of 47 years old(24-74),including 12 males and 9 females,.All the patients were diagnosed according to WHO criteria.1.3 Cell line:Chronic myelogenous leukemia cell line-K562 cell was kinderly provided by Stem Cell Research Center of Zhengzhou University.1.4 Controls:Total 10 patients with non-malignant hematological disease were used as controls.2.RT-PCR:Total RNA was extracted from bone marrow mononuclear cell (BMMNC) by RNAiso reagent.The mRNA expressions of CDX2 in BMMNC were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).The electrophoresis of 5μl PCR products was performed at 80V for 35 min in 2.0%agarose gel containing 0.5 mg/l ethidium bromide.And the bands were viewed and photographed under UV illumination.The transcripts were estimated by the optical density ratio of CDX2 toβ-actin.3.Statistical analysis.Software SPSS 13.0 was used to analyze data.Differences between groups were evaluated by the independent samples test and ANOVA. Correlation was analyzed by Pearson correlation.P values of less than 0.05 were considered statistically significant.Results1.Expression of CDX2 mRNA in acute leukemiaThe expression of CDX2 mRNA in 44 cases of de novo acute leukemia patients had the positive rate of 90.9%and the average level of expression with 0.75±0.16. There was no expression of CDX2 mRNA in 10 control cases,with expression level 0. the difference of CDX2 mRNA expression between two groups were statistically significant(P＜0.05).The expression rate of CDX2 mRNA in acute myeloid leukemia(AML) and acute lymphocytic leukemia(ALL) were 90.6%and 91.7%,respectively.The average expression level were 0.73±0.17 and 0.84±0.16,respectively.The different expression of CDX2 mRNA in two groups were not statistically significant(P＞0.05).The average expression level of CDX2 mRNA in de novo group,relapse group and CR group were 0.75±0.16,0.71±0.14 and0.00±0.00,respectively.There was no statistically significant difference between relapsed and de novo group(P＞0.05). Compared to CR group,de novo group and relapse group expressed significantly higher level of CDX2 mRNA(P＜0.05).2.Expression of CDX2 mRNA in chronic myelogenous leukemiaThe positive expression rate of CDX2 mRNA in 21 cases of chronic myelogenous leukemia patients were 66.7%with an average expression level of 0.75±0.15.Compared to control group,the diffrerence was significant(P＜0.05).The average expression level of CDX2 mRNA in CML-CP,CML-AP and CML-BP were 0.58±0.32,0.71±0.20 and 0.78±0.45,respectively.Compared to CML-CP, CML-AP and CML-BP expressed significantly higher level of CDX2 mRNA(P＜0.05).There was no expression of CDX2 mRNA in 1/4 of accelerated phase patients and 6/13 chronic phase patients.3.Expression of CDX2 mRNA in chronic myelogenous leukemia cell line-K562 cell:Expression of CDX2 mRNA K562 cell is the CDX2 mRNA was detected by RT-PCR with an average expression level of 0.77±0.37,experiments repeated 15 times.Compared to CML-AP and CML-BP,the difference of CDX2 mRNA expression had no statistical significance(P＞0.05).4.Relationship of CDX2 mRNA expression and blasts in bone marrow:The expression level of CDX2 mRNA was positively correlated with the count of leukemic blasts in bone marrow from acute leukemia patients and chronic myelogenous leukemia patients(r=0.781,r=0.357,P＜0.01).5.Expression of CDX2 mRNA with leukaemia patient clinical features.There is no significant difference between the expression of CDX2 mRNA and patients gender,age,white blood cell count and extramedullary infiltration(P＞0.05).Conclusions1.The CDX2 mRNA is up-regulated in de novo and relapsed acute leukemia patients,and down-regulated after patients reached CR,indicating that CDX2 may contribute to the genesis and development of acute leukemia.2.There is no difference of expression of CDX2 mRNA between subtypes of acute leukemia. 3.The CDX2 mRNA expression levels in part of patients with chronic granulocyte leukemia are up-regulated,especially in CML-AP and CML-BP.K562 cell also expressed high level of CDX2 mRNA,suggesting that CDX2 may involved in the genesis and development of leukemia.4.There is a positive relationship between mRNA expreesion of CDX2 and the count of leukemic blasts in bone marrow,demonstrating that CDX2 mRNA is expressed by leukemic blasts. |
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