The Expression And SNP Analysis Of BRD7 Gene In Leukemia Cells And Effects On The Cell Cycle And Cell Apoptosis

[Background]Leukemia is a group of malignant clone's diseases of hematopoietic stem sells,and is also one of frequent teen-age malignant cancers.The incidence rate of the cancer has the tendency of step-up recent years.An understanding of the molecular pathogenesis of Leukemia should be very important to defend and treat this cancer. Leukemia is the consequence of a multistep process resulting from a series of genetic changes and incidence,and its process is relate with activation of oncogene or inactivation of antioncogene.The classical example of proto-oncogene activating carcinogenesis is that the characteristic fusion gene 'bcr-abl' in chronic granulocytic leukemia which is formed by Philadephia chromosome translocation,t(9;22) (q34;q11).The fusion mRNA codes the abnormal tyrosine kinase protein p²¹⁰that has enhanced activity compared to the wild type tyrosine kinases.Untimately,it induce the cell cancerrization.With the accumulation of experimental indicator on Leukemia,many novel genes are along with Leukemia and abnormal,such as gene mutations,gene deletions,gene insertions,gene overexpression and inactivating function and so on.In these genes,some genes as RAS,MYC,p16 in leukemia have the same characteristic as solid tumors,but the others represent different expression patterns and pathogenesis,p53 is mutated in approximately 60%of all solid tumors and it is the main reason that p53 has low expression and loss of the function of suppressing tumor.In contrast,p53 has high protein expression and low frequent mutation (＜20%)in most patients with leukemia.Perhaps there is no connection between the gene inactivating function and itself mutation.Although many genes' function and machanisms in Leukemia has been explicit,all novel gene can not explain all pathogenesis of all Leukemia.Thereby,It has become one of the hotspots to investigate Leukemia by identifying new correlative oncogene or tumor suppressor genes and studying its biologic function.BRD7 is a tumor suppressor gene of nasopharyngeal carcinoma (NPC)cloned by cDNA representational differential assay in molecular genetics laboratory of Cancer Reasearch Institute of Central South University.The full-length of BRD7 cDNA is 2317bp(genebank accession number:AF152604)and locates 16q 12.1-12.2.its open reading frame(ORF)includes 651 amino acids.The past research indicated that decreased expression of BRD7 was detected in NPC biopsies and HNE1 cells.After the gene is transfection into HNE1 cell,The overexpressed BRD7 can inhibit the proliferation of NPC and arrest them in G0/G1 phase by transcriptionally regulating some important molecules involved in Ras/MEK/ERK and Rb/E2F pathways.At the same time,the protein of BRD7 locates at HNE1 karyon and interact with nuclear transcription factor IRF2 and adenovirus nuclear protein E1B-AP5 to affect their transcription activity.[Methods]In order to analyze the change of BRD7 expression and the correlation between the BRD7 expression and the clinical character of patients with acute leukemia,RT-PCR,immunohistiochemical stains, Western blotting were used to detect the BRD7 expression in acute leukemias and leukemic cell lines.Single-Strand Conformation Polymorphism(PCR-SSCP)and DNA sequence analysis also were used to identified BRD7 mutation or single nucleotide polymorphism(SNP) to investigate the relation of BRD7 and acute leukemia.The BRD7-expressing K562 cells were constructed by electroporation.The cell was used to study the effect of BRD7 on cell cycle and apoptosis through fluorescence protein and Flow Cytometry assays.[Results]Partâ… The Expression and Clinical Signification of BRD7 gene in Leukemia Cells1.The mRNA expression of BRD7 in patients with acute leukemiaBone marrow mononuclear cells expressed a 259bp cDNA band of RT-PCR product in 52 patients with acute leukemia and 32 normal subjects,that indicated BRD7 was expressed not only in leukemia cells but also in normal Bone marrow cells.Compared with inter contrast GAPDH,there was no significant difference in acute lymphoblastic leukemia(ALL)and acute myelogenous leukemia(AML) (P＞0.05).However the relative expression BRD7 in patients with acute leukemia was higher than in the control,and the difference was significant(t=3.672,P=0.001;t=2.148,P=0.037)2.The protein expression of BRD7 in patients with acute leukemiaBRD7 positive cells in Bone marrow mononuclear cells from 7 patients with acute leukemia and 3 normal controls were assayed by immunohistiochemical stains.The result showed there were high percentage of BRD7 protein positive cells in patients with acute leukemia. 3.The correction about BRD7 with WBC counts and platelet counts in patients with leukemiaBy correlative analysis of Kendall's tau-b,the mRNA expressions of BRD7 gene in 52 patients with leukemia at onset were not correlated with white blood cells(WBC)counts in total peripheral blood (r=0.086,P=0.376),and the same as platelet counts(r=0.011,P=0.912).4.The mRNA expression of BRD7 in hematological malignancies cell linesRT-PCR was used to detect the mRNA expression of BRD7 in hematological malignancies cell lines,we found these cell lines definitely express BRDT,like human Leukemia cell K562,HL-60,Jurkat cell, human multiple myeloma cell KM3,human non-Hodgkin lymphoma cell Raji and human nomal lymphocyte B958,but the expression of BRD7 in K562 was low.5.The protein expression of BRD7 in hematological malignancies cell linesBRD7 protein always was express in human Leukemia cell K562, HL-60,Jurkat,human multiple myeloma cell KM3,human non-Hodgkin lumphoma cell Raji and human nomal lymphocyte B958,the BRD7 protein level in K562 cells was also relative low.Partâ…¡Single-Strand Conformation Polymorphisms in BRD7 Gene1.PCR-SSCP were used to filter BRD7 gene mutationThe abnormal removing band was detected by PAGE techniques in some samples from 30 normal controls,52 patients with leukemia and 30 normal marrow subjects.That indicated the 440-840^thbase possibly have mutation or polymorphism.2.The SNP genotype of BRD7 identified by DNA sequencing analysis Three SNPs(C657A,C495T and A737G)in BRD7 gene coding region(440-840bp)were found.One of those cSNPs is C657A transition,which creates a missense change of Asp 181Lys.Another SNP (C495T)is synonymous polymorphism which was coupled interestingly with the A737G alteration.The third SNP(A737G)is non-synonymous polymorphism which results in a change of Glu208Gly.3.The frequency distribution among leukemias and controls with genotypic variants of SNPs 737A/G,495C/T and 657C/AAt the gene position of A737G,BRD7 showed three genotypes, AA,AG and GG.The genotype frequencies in leukemias group were AA.11.5%,AG:32.7%,GG:55.8%;the allele frequencies of A and G were 27.9%and 72.1%respectively.While in controls the genotype frequencies were AA:38.4%,AG:28.3%,GG:33.3%with A and G allele frequencies of 53.4%and 46.6%respectively,the genotype and the allele frequencies in both leukemias and the control group were significantly different(X²=25.76,P＜0.01).Compared with the genotype(AA), individuals with AG genotype and GG genotype had higher leukemic risk (OR=1.12,95%CI:1.02～1.23;OR=1.23,95%CI:0.55～2.77).C495T was coupled with A737G,its genotype frequencies and distribution was similar to A737G.The genotype frequencies C657A had no difference in the two groups.4.The comparison about the mRNA expression of BRD7 of three genotypes SNP 737A/G in acute leukemiasAmong the three genotypes of A737G in acute leukemias,GG which compared to inter contrast GAPDH had the highest level of 0.512±0.192, the AG had the lowest level of 0.467±0.108.Statistical analysis showed there was no significant discrepancy among the mRNA expression levels of AA,AG and GG genotypes in leukemias group(P＞0.05).Partâ…¢The effect of BRD7 on cell cycle and apoptosis in leukemia cells1.The subcellular localization of BRD7 in K562 cell lineAfter the K562 cells were transfected by electroporation expressing pEGFP-C2/BRD7,GFP direct fluorescence assays were performed to detect the subcellular localization.The result consistently showed BRD7 in K562 was localized in nucleus with a diffused or stripped pattern.It was also shown that BRD7 distribution in nucleus was similar to that of chromatin.2.The effect of BRD7 to cell cycle and apoptosis in leukemia cellsAfter the leukemic cell line K562 were transfected by electroporation expressing pEGFP-C2/BRD7 or blank pEGFP-C2 vector without BRD7,Flow Cytometry assays was performed,and the result showed BRD7 could not only partly block cell cycle G0/G1→S phase, but also obviously initiate cell apoptosis.[Conclusions]1.BRD7 was expressed in bone marrow mononuclear cells from patients with acute leukemia and normal subjects,BRD7 was up-regulated in acute Leukemia cells.2.BRD7 participates in the formation of acute leukemia,but the mRNA expression level of BRD7 gene was not correlated with the WBC counts and platelet counts in patients with acute leukemia at onset.3.All the cell lines of malignant cancers in blood system like K562,HL-60,Jurkat,KM3,Raji and normal lymphocyte B958 can express BRD7,but the expression of BRD7 in K562 was low.4.BRD7 gene(440-840bp)included three coding region SNPs(C657A, C495T and A737G),and A737G was coupled interestingly with the C495T.The mutant allele frequencies of the two coupled SNPs in patients with acute Leukemia was significantly higher than the controls, that indicated SNPs may be one of acute leukemia genetic susceptibility factors.5.BRD7 protein in K562 cells was localized in nucleus,also it has double function in blocking cell cycle and initiating cell apoptosis.