Distinctive MicroRNA Expression In Myelodysplastic Syndromes

Background:Myelodysplastic syndromes(MDS)is a clonal malignant disease of the haematopoietic stem cell(HSC).MDS comprises a heterogeneous group that share a high frequency of recurrent chromosomal aberrations,high risk of transformation into secondary acute myeloid leukemia,and exhibit blood cytopenias,ineffective hematopoiesis and usually hypercellular or normocellular bone marrow(BM).According to epidemiology of MDS,over 50 year old people were the prominent population occupied about 50%-70%cases.Nowadays,morbidity of MDS trends to raised with the aging of population in China.Thus,how to early diagnosis and early treatment,how to evaluate the risk of transformation is important for improving prognosis and prolonging life-span.Our study is to explore the pathogenesis of MDS and find some prognosis index which can predict MDS transforming to leukemia.MDS is a patho-procedure involving multigene and multisegment with high heterogeneity,and we know little about the initial factors and molecular mechanism of MDS.From clinical research of MDS,over 70%MDS patients existing cytogenetic disorder or gene mutation which was thought as main molecular mechanism of MDS.Meanwhile,signal transduction pathway,cell cycle,transcription factor and so on,all involve in the progress of MDS.However,what reason contribute partly MDS individuals to AML?Research found that many genes expressed distinctively with different risk and condition in MDS.It's not yet clear that which gene mutation is the initial factor,which gene disorder with disease progression,and whether the abnormal genes interaction each other or not.MicroRNA(miRNA)is a kind of gene regulation RNA that caused widespread concern in recent years.miRNA is a class of endogenous non-coding RNA in length from 20 to 22 nucleotides.By pairing the 3'-UTR region of target gene in a complete or incomplete way,miRNA can degrade target gene mRNA or inhibit translation of target gene expression to achieve the regulation at the level of transcription.miRNA may inhibit the extension or termination of translation,or degradation of newly synthesized ribosome nascent chain.Thus,miRNA play an important role to regulate gene expression.With the depth of miRNA research,it was found that widely exist in small biological molecules each species of organism development,differentiation,proliferation,apoptosis,immune regulation and other physical activities as well as the incidence of malignant tumors have regulatory role.In recent years,miRNA expression profiling has shown that miRNAs are associated with the development,progression and prognosis of several diseases,indicating that miRNAs expression profiling can serve as the biomarkers of diagnosis and treatment for diseases.Evidences showed that miRNAs regulate the directional differentiation potential of HSC.As the non-specific regulatory factors,miRNAs may play important role in the molecular regulation of diseases with HSC damaged.MDS is a clonal malignant disease of HSC or pluripotent stem cell,the multiple-gene change is critical to the pathogenesis of MDS.Considering the important role of miRNAs in the pathogenesis of leukaemia,we hypothesis that miRNAs may play important role in the development and prognosis of MDS.However,little is known about the role of miRNAs in the pathogenesis of MDS currently.Part of MDS patients with a high risk of transformation to AML,based on clinical manifestations and laboratory findings,it can be carried out in different risk groups.In the treatment of high-risk patients,doctors are often tend to be more aggressive treatment,but the effect is not satisfactory.Therefore,we will focus on the research on the patients of high-risk MDS to AML transformation,we hope that through the study of miRNA in the pathogenesis of these special populations,we will explore specific miRNA that can early prediction of disease change,and provide new methods for these patients.In this study,microRNA microarray technology is used to screen out differential expression miRNA profile in patients with MDS and MDS transformed to AML(t-MDS).The differential expression miRNA are selected and validated by real-time PCR technology in large sample of MDS and t-MDS.We next employ bioinformatics techniques to predict the target genes of these miRNA and analyzed and their signaling pathways in the pathogenesis of MDS.Part of differential expression miRNAs are selected for further functional studies.We used the miRNA mimics and inhibitors to up-regulated and down-regulated miRNA in MDS cell lines and observe the cell growth and apoptis,we also detect the expression of miRNA target genes to confirm wether they regulate the development and progression in MDS.This study has important theoretical significance to elucidate the pathogenesis of MDS,and is expected to provide a scientific basis for finding new therapeutic targets for MDS.Objective:By screening differential expression miRNA profile in patients with MDS and t-MDS,this study will find the related miRNAs for MDS and t-MDS,and analyze the relationship between miRNA expression and clinical features of MDS.The significantly differentially expressed miRNAs are selected and changed their expression,and the growth and apoptis of MDS cell lines were observed.This study will explore specific expression the mechanisms of miRNA on MDS transformed to AML at the cellular level.Materials and Methods:In order to explore miRNA expression profile in MDS,we used a novel miRNA-specific microarray technology and Hierarchical Cluster Analysis,and the results were verified by real-time PCR.After enlarging the sample to test the aberrant expression of miRNA by Real-time PCR in MDS,t-MDS and controls.It included 54 MDS(32 low risk+poor risk-land 22 poor risk-2+high risk),16 t-MDS and 19 healthy controls.Meanwhile we analyzed the correlation between the expression of specific miRNA and the clinical features of the patients.Target genes of the specifically expressed miRNA were predicated by Bioinfor-matics technology,and we chose some potential genes to validate their expressions in MDS and t-MDS by real-time PCR.We transferred miR-196b-5p mimics and inhibitor into human MDS-L cells to regulate the expression of miRNA,then estimated the functions of miRNA at cell level and observed the proliferation,apoptosis of the cells seperately.Results:We obtained 367 miRNA which expressed differently between MDS and normal.Among them,19 miRNA had significantly different expression(Fold-change>5):10 miRNA were up-regulated and 9 were down-regulated.MiR-196b-5p showed remarkable up-regulation in MDS,especially in poor risk-2 MDS and high risk MDS,and its level is even more higher in t-MDS.(P<0.05).We speculate that miR-196b-5p may dedicate to the occurrence of MDS and the transformation of MDS to AML.125 target genes of miR-196b-5p were predicted by Bioinformatics,heir known functions,and we chose 6 target genes from them according to their function,which related to proliferation and apoptosis of hematopoietic cells.These genes including ANXA1?DICER1?BIRC6?FAS and HOXC8.Their expression levels were tested in MDS,t-MDS and controls respectively.DICER1 and ANXA1 showed down-regulated tendency corresponding to the up-regulation of miR-196b-5p,according to the negative regulatory mechanism.Using mimics and inhibitor of miR-196b-5p,we constructed vector to transfer them into ·MDS-L cells.The result showed that after up-regulating the expression of miR-196b-5p,the rate of cell growth and differentiation speeded up and apoptosis decreased.On the contrary,when the expression of miR-196b was down-regulated,the rate of cell growth and differentiation slowed down and apoptosis increased.Conclusion:This study indicated that the specific miRNA profile in MDS may be a new biomarker to this disease.miR-196b-5p may take part in the transformation of MDS to AML by regulating its target gene-DICER1.miR-196b-5p is prospective to be an early intervention spot of MDS which have high risk of translating to AML.