| PrefaceFA is an autosomal recessive disease characterized by chromosome instability,cellular hypersensitivity to DNA cross-linking agents such as Mitomycin C (MMC) and Cisplatin,and increased predisposition to cancers,mainly leukemias and squamous cell carcinomas of the head and neck or gynecologic system.FA can be divided into at least 13 complementation groups(A,B,C,D1,D2,E,F,G,I,J,L,M and N),and 12of the 13 responsible FA-associated genes have been identified so far,the products of which function in the FA/BRCA pathway.A key event in the pathway is the monoubiquitination of the FANCD2 protein,which depends on a multiprotein FA core complex.FANCF is an essential component of the Fanconi anemia(FA) complementation groups.It acts as a flexible adaptor protein that plays a key role in stabilizing the FA complex formation and the monoubiquitination of FANCD2.So FANCF is an important protein that maintains the function of FA/BRCA signaling pathway which may have function in the several cellular processes,including cell cycle control,apoptosis,DNA repair,gene transcription and stability.It is reported that low expression and dysfunction of FANCF protein were related to FANCF promoter hypermethylation.At present,It has been confirmed that FANCF is related to development of many kinds of tumors(ovarian caner,oral caner,lung canr and andcervial caner).But the study on AML and MDS is rare.The purpose of the study is to establish base for gene therapy through comfirming the relationshipof FANCF,FANCD2 protien and FANCF methylation.Approach for the relationship of pathogenesy with prognosis of AML and MDS.Hope to find a new strategy for therapy.Materials and methods1.The study objet:111 AML patients of CMU OPD and WD science September 2007 to July 2008,containing 62 men,49 female.M-F is1.2:l,MS-age is 56,preliminary diagnosis 64,complete remission(CR)27,relapse group 20;46 MDS patients,low-risk group has 18 specimens(RA 8,RAS 10),high-risk group has 28 specimens(RAEB 13,RAEB-T 15).MS-age is 67,M-F is 1:1,control group has 42 patients,which are megaloblastic anemia,hypoferric anemia,or thrombocytolytic anemia.diagnosis are consistent with FAB diagnosis.2.Method:(1) Mono-nucleus cell are exact from bone marrow by Fioll density gradient centrifugation.(2) Exact DNA from cell with Phenol-chloroform-Phenol centrifugalization.(3) MS-PCRwas used to detect the methylation of FANCF.(4) Detect FANCF/FANCD2 protein by histochemical stain.(5) Detect FANCF/FANCD2 protein by Western blot.(6) Statistic Analyze with SPSS 13.0,rank sum test for measurrement data,X~2-test for enumeration data,Descriptive analyses adopt median(M).Result1.The methylation of the FANCF in AMLHyper-methylation was found in group of patients with AML,there was statistical significance between AML group and control group(P<0.05).Preliminary diagnosis group show higher methylation of FANCF than patients of CR.There was statistical significance in group comparison(P<0.05).There is no visible different between relapsed group and preliminary diagnosis group(P>0.05),but there is visible different between relapsed group and CR group(P<0.05).Comparing CR group with control group there was no statistically significant(P>0.05).2.The expression of FANCF,FANCD2 protein in AMLHistochemical stain show that the expression of FANCF protein in preliminary diagnosis group is 9.3%,CR group is 74.1%,relapse group is 10.0%,control group is 80.9%;Western blot show that the expression of FANCF protein in preliminary diagnosis group is 31.2%,CR group is 77.8%,relapse group is 30.0%,control group is 95.2%,It's significantly lower in preliminary diagnosis than in control group(P<0.05), comparing preliminary diagnosis group,CR group has statistically significant(P<0.05),there is obvious different between relapse group and CR group,Comparing with CR group with control group has no statistically significant(P>0.05).Histochemical stain show that the expression of FANCD2 protein in preliminary diagnosis group is 6.3%,CR group is 63.0%,relapse group is 5.0%,control group is 71.4%;Western blot show that the expression of FANCD2 protein in preliminary diagnosis group is 34.3%,relapse group is 35.0%,CR group is 74.1%,control group is 88.1%,It's significantly lower in preliminary diagnosis than in control group(P<0.05),comparing preliminary diagnosis group,CR group has statistically significant(P<0.05);there is obvious different between relapse group and CR group(P<0.05);Comparing with CR group with control group has no statistically significant(P>0.05).3.The methylation of the FANCF in MDSBoth high-risk and low-risk MDS show high-methylation of FANCF.There was statistical significance between MDS group with control group(P<0.05),as well as between high-risk MDS and low-risk MDS(P<0.05).4.The expression of FANCF,FANCD2 protein in MDSHistochemical stain show that the expression of FANCF protein in low-risk group is 5.6%,high-risk group is 3.6%,control group is 80.9%;Western blot show that the expression of FANCF protein in low-risk group is 11.1%,high-risk group is 7.1%, control group is 95.2%,comparison has statistically significant(P<0.05).Histochemical stain show that the expression of FANCD2 protein in low-risk group is 16.7%, high-risk group is 7.1%,control group is 71.4%;Western blot show that the expression of FANCD2 protein in low-risk group is 22.2%,high-risk group is 10.7%,control group is 88.1%,comparison has statistically significant(P<0.05).ConclutionThe experiment demonstrate that FANCF in patients with AML showed hyper-methylated,as well as methylation of FANCF is obviously higher in preliminary diagnosis group than CR group,which demonstrates that FANCF gene probably plays a role in the pathogenesis of AML.The preliminary diagnosis and relapse groups of AML show obviousely low-expression of FANCF protein than control group,which elucidates that FANCF protein probably have a relationship with the pathogenesis and prognosis of AML.Methylation of FANCF is obviously higher in high-risk MDS than low-risk MDS,which demonstrates that FANCF gene probably plays a role in the pathogenesis of MDS.the different between high-risk/low-risk group of MDS and control group indicatings that FANCF pretein is concerned with pathogenesis of MDS.The expression of FANCD2 protein is consistent with FANCF. |
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