Detection Of HMGA2 In Patients With Chronic Myeloid Leukaemia Using A Quantitative Real-time PCR Method

Background and objective:HMGA2,which belongs to high mobility group family,is a small nuclear chromatin associated protein.The member of HMG family have the characteristics of high mobility in PAGE because of small molecular weight.There are three short basic repeats,the so-called AT-hooks,located in the amino-terminal region of the protein, binding the minor groove of AT-rich DNA sequences.HMGA2 protein does not have transcriptional activity per se.However,by binding the DNA sequences,they alter chromatin structure,acting as an architectural protein,and thereby orchestrate the assembly of nucleoprotein complexes involved in gene transcription and regulate the transcriptional activity of several genes through a complex network of protein-DNA and protein-protein interactions.HMGA2 plays an important role in growth and development.HMGA2 is widely expressed during embryogenesis,whereas their expression is absent in most of the normal adult tissues.Many studies have demonstrated the misexpression,such as overexpression and rearrangement,of the HMGA2 gene in benign and malignant neoplasias,included hematopioetic malignancies.Several studies indicate that HMGA2 protein has oncogenic activities,being causally involved in neoplastic transformation,and blockage of their synthesis suppresses the malignant phenotype. For example,HMGA2 was not present in retrovirally infected rat thyroid cells that did not have a fully malignant phenotype,whereas they were constantly present in those cells that,following retroviral infection,underwent a full malignant transformation;expression of either a full-length or a truncated human HMGA2 transgene in mice,under the control of the differentiated mesenchymal cell-specific promoter,induces the onset of different benign turnouts of the mesenchymal tissue; when normal thyroid ceils(FRTLS) were transfected with an antisense construct against HMGA2 and then infected with transforming myeloproliferative sarcoma virus or Kirsten murine sarcoma virus,in contrast with untransfected cells or cells transfected with the sense construct,those containing the antisense construct did not grow in soft agar or form turnouts in athymic mice.Several mechanisms have been proposed to account for the transforming ability of the HMGA2 protein.①HMGA2 protein inhibits the activation of DNA repair systems,such as binds the promoter of the nucleotide excision-repair gene ERCC1 and negatively modulates its activity.②HMGA2 protein may interfere with the normal cell cycle through multiple ways.HMGA2 protein can bind to pRB and displace histone deacetylase 1 from the pRB-E2F1 complex,resulting in enhanced acetylation of both E2F1 and DNA-associated histones,thereby promoting E2F1 activation and cells proliferation.③HMGA2 proteins induce tumorigenesis by modulating the epithelial-mesenchymal transition(EMT).HMGA2 can have a role in EMT comes from recent studies demonstrating that transforming growth factorβmediates EMT by inducing HMGA2 through the Smad pathway④The chromosomal rearrangement leads to overexpression of truncated-HMGA2 protein without the 3' untranslated region(UTR).Truncated-HMGA2 proteins lose their multiple target sites in the 3'UTR,so can not contact with microRNAs included let-7 and lose the inhibition of microRNAs.This disrupted repression induces tumorigenesis finally.There is less research on hematologic neoplasm,some scholars checked CD34 positive hematopoietic stem cells and their normal and malignant descendants for HMGA2 expression.CD34 positive stem cells from healthy donors and the leukemia samples tested were positive while all peripheral blood samples from healthy volunteers were negative,they have concluded that the expression of the HMGA2 gene in leukemia seems to be a secondary effect due to abnormal stem cell proliferation and might be a sensitive tumor marker for particular types of leukemia. HMGA2 rearrangements,following translocation involving the chromosome band 12q13-15,have been reported in patients of Richter transformation of a chronic lymphocytic leukaemia,acute lymphoblastoid leukaemia myelofibrosis with myeloid metaplasia,myelodisplastic syndrome and acute myeloid leukaemia.FISH analysis showed that the rearrangements concerning HMGA2.It is interesting to note that most of the patients showing HMGA2 rearrangements did not have a long survival, suggesting that HMGA2 expression might indicate poor prognosis.Recently,Meyer first study the HMGA2 expression in blood from CML patients compared to blood from healthy donors presenting quantitative analysis.HMGA2 expression is higher in a case of CML-AP and a case of CML-BP.Chronic myeloid leukemia(CML) is a hematopoietic disorder characterized by the malignant expansion of pluripotential stem cells.Philadelphia chromosome(Ph) and Bcr/Abl fusion gene are malignant clonal markers of CML.P210 protein,encoded by Bcr/Abl fusion gene,with a deregulated tyrosine kinase(PTK) activity,which induces phosphorylation of a series of downstream signal molecules is identified as having a central role in the pathogenesis of CML.The courses of CML include chronic phase(CP),accelerated phase(AP) and blastic phase(BP).The treatment reaction of patients in AP and BP is usually poor.The blast crisis of CML is often accompanied with amplification of bcr/abl fusion gene and enhanced activity of PTK. The mechanisms of transformation to BC are varied and not entirely understood.So far the best characterized include differentiation arrest,genomic instability,telomere shortening and loss of tumour-suppressor functions.To explore the relationship between the progression of CML and HMGA2,we select the peripheral blood of patients with CML during different periods,and apply a real-time PCR for relatively quantitative detection of HMGA2 by TaqMan probe.Methods:1.Investigation objectNormal peripheral blood(totol 5 samples) were from the laboratory department of NanFang hospital,CML-CP peripheral blood(totol 12 samples) and CML-AP/BP peripheral blood(totol 12 samples) were from department of hematology of NanFang hospital.2.Experimental method1) Morphologic examination of bone marrow and peripheral blood;karyotype analysis.2) Analysis the level of bcr/abl fusion gene by FISH.3) Detection of HMGA2 expression by real-time fluorescent quantitative PCR.①Design and synthesis of primers and taqman probes of HMGA2 and internal standard GAPDH respectively.②Collection of the peripheral blood of patients and extraction of the mononuclear cells of whole blood.③Isolation of total RNA.④Reverse transcription PCR.⑤PCR,construction of HMGA2 standard plasmid. ⑥Detection of HMGA2 using a real-time quantitative PCR method.4) tatistics analysisThe rank sum test was used to assess the HMGA2 expression difference between CML-CP and CML-AP/BP.Correlation analyses were done using Spearman's correlation test to explore correlation between HMGA2 expression and blast cells or bcr/abl expression respectively,p values below 0.05 were considered significant.Results:1.Low transcript levels were measured in patients in chronic phase,and elevated levels were detected in AP/BP.The difference was statistically significant(Z=-4.157,P＜0.01).2.There were no significant correlations between bcr/abl and HMGA2 transcript levels for all CML patients or CML-AP/BP patients(rs=0.078;p=0.471/ rs=0.058;P=0.736).3.There were significant correlations between blast cells count and HMGA2 transcript levels for CML-AP/BP patients(rs=0.636;p=0.017).There were no significant correlations between HMGA2 transcript levels and leukocyte count (r_s=-0.067;P=0.654).Conclusions:1.The transcript level of HMGA2 is related to the stages of CML,which is significantly increased in AP/BP.HMGA2 maybe plays an important role in the development of disease process.The mechanism needs further research.2.There are no significant correlations between bcr/abl and HMGA2 transcript levels,which may suggest that bcr/abl fusion gene and HMGA2 gene play its role through different ways in the development of disease process. 3.The transcript levels of HMGA2 positively correlated with proliferation of blast cells.HMGA2 expression may correlated to the leukaemic cells accumulating during progression of chronic phase to blast crisis.The clinical applications such as monitoring the progress of disease,observing the therapetic results and judging prognosis need further research.Innovative points:Relative quantitative analysis of HMGA2 mRNA by real-time fluorescent quantitative PCR in CML have not been reported previously.We get some new concludes:The transcript level of HMGA2 is significantly increased in AP/BP,there are no significant correlations between bcr/abl and HMGA2 transcript levels,the transcript levels of HMGA2 positively correlated with proliferation of blast cells.