The Expression,mutation Spectrum And Mechanism Of FAMLF And Its Intronic MiR-181a1/b1 In Acute Myeloid Leukemia

Background:Acute myeloid leukemia(AML)is a group of hematologic malignancies,which is derived from clonal hematopoietic stem cells.There is a high degree of heterogeneity in the Clinical features,efficacy,and prognosis between different subtypes of AML patients,which is closely related to the highly heterogeneous of molecular genetics abnormalities among various AML subtypes.The precise pathogenesis is still unknown.So discover new genes associated with AML and further study its role in the pathogenesis of AML is still an important issue in nowadays medical research.Familial acute myeloid leukemia has unique research value.The genetic abnormalities associated with familial AML can be extended to the sporadic AML,and help to elucidate the pathogenesis of AML.We have successfully cloned a full-length cDNA of a novel gene related to AML,named Homo sapiens familial acute myelogenous leukemia related factor.With bioinformatics analysis,we reveal the existence of at least three splice variants,named as FAMLF-1,FAMLF-2 and FAMLF-3,and also find miR181a1/b1 in the second intron regions of FAMLF gene family.The expression of FAMLF-1,FAMLF-2 is significantly higher in de novo AML peripheral blood mononuclear cells than normal control.So we speculate FAMLF-1 may play a role in the development of AML.To further explore the function of FAMLF splice variants in AML,we intends to further validate FAMLF-1 mRNA full length,define whether FAMLF-1 encodes a protein,improve the correlation analysis between the expression of FAMLF-1with the clinical Hematology characteristics of patients with AML,further analysis the correlation expression of miR181a1 and FAMLF-1 in AML patients,analysis the relation between FAMLF gene family mutation spectrum and its clinical significance in patients with AML,downregulate the expression of FAMLF-1 at the cellular level in order to explore its function in AML.ObjectiveAnalysis FAMLF-1 gene expression,mutation and its clinical significance in AML patients.Reveal the role of FAMLF-1 gene in the pathogenesis of AML.MethodsWe defined the full length of FAMLF-1 with Northern blot.We predicted and synthesized polypeptide of FAMLF-1 used to produce antibodies,used western blot assays to evaluate the open reading frame of FAMLF-1.We determined the expression of FAMLF-1 in AML patients using real-time quantitative polymerase chain reaction(RQ-PCR)with SYBR Green,analyze the relationship between its expression levels and clinical hematology characteristics.We detected the expression of miR-181a1 in AML using real-time quantitative PCR with Taqman probe,analyzed the correlation of expression levels between miR-181a1 and FAMLF-1.Using Sanger sequencing method,we depicted the mutations of FAMLF gene family’s exons and miR-181a1 / b1 in AML patients,analyzed the clinical significance of mutation.Then,RNA interference to silence FAMLF-1 gene expression was applied to observe biological changes of Kasumi-1 in order to explore it’s molecular mechanism.Results1.Northern blot confirmed the full-length of FAM LF-1 gene as 2.3 kb,the gene was highly expressed in AML patients and cell lines,expressed at lower level in the normal control group.Western blot analysis confirmed that the molecular weight of FAMLF-1 was about 8kd,the protein is highly expressed in patients and cell lines of AML,expressed at lower level in the normal control group.2.RQ-PCR showed that FAMLF-1 mRNA expression in AML patients was significantly higher than in the normal individuals(p<0.0001).FAMLF expression of M2 subtypes was higher than that of non-M2 subtypes(p=0.0152).3.The expression of FAMLF-1 was positively correlated with hemoglobin levels,percentage of peripheral blood blasts and serum HBDH concentration in patients with AML(p<0.05).Moreover the expression of FAMLF-1 was positively correlated with hemoglobin levels,white blood cell count,percentage of peripheral blood blasts and serum HBDH concentration in patients with M2(p<0.05),but not in patient with non-M2(p>0.05).4.AML patients with higher FAMLF-1 expression had significantly higher complete remission rate but lower relapse-free survival and longer forward overall survival(p<0.05).M2 patients with higher FAMLF-1 expression had significantly higher complete remission rate but not non-M2 patients.5.There was also a positive correlation between the expression of FAMLF-1 and miR-181a1(p<0.05).6.Genotyping and association analysis showed that the GTAGG haploid of FAMLF gene family increased the prevalence of M2(OR=1.92.3,95%CI: 1.0201-3.6250,P=0.0419).7.The pGV248/GFP-FAMLF-1 RNAi expression plasmids targeting on FAMLF-1mRNA were successfully constructed.8.FAMLF-1-RNAi-LV was successfully transfected into Kasumi-1 cells.The efficiency of FAMLF-1 mRNA knockdown was about 70% by real-time quantitative RT-PCR.Which showed that cell model of FAMLF-1 gene silencing were successfully constructed.9.Growth curve,colony formation assay,flow cytometry,real-time quantitative PCR,and Western blot were used to access the effects of FAMLF-1 RNAi on Kasumi-1cell.It showed that FAMLF-1 gene silencing could result in many cellular bio-behavior changes in Kasumi-1,such as inhibited the proliferation,arrested cell cycle in G0/G1 phase,and promote differentiation,which may be related to Akt pathway.Conclusions:1.FAMLF-1 gene was 2313 bp in length,encoding about 8kd protein.FAMLF-1was obviously over-expressed in patients with AML,especially in M2.Higher expression of FAMLF-1 was related to haematological characteristics in patients with AML especially in M2.The GTAGG haploid of FAMLF gene family could increased M2 risk.2.Knockdown of FAMLF-1 could inhibit cell proliferation,inhibited of G0/G1 phase process and promote differentiation in Kasumi-1 cells,may be involved in the pathogenesis of AML through Akt pathway.