A Preliminary Study On The Expression Profile Of Exosomal MicroRNA In Acute Myeloid Leukemia Patient

Objective: Acute myeloid leukemia(AML)is a malignant clone haemopoiteic stem cell disease.Existing diagnostic method based on morphological characteristics of blood cells in bone marrow is not helpful for early disease detection and the diagnosis repeatability is poor.A large amount of studies have found that,micro RNA(mi RNA or mi R),enriched in exosomes with good stability thanks to the protection from its surface film,can be an ideal molecular marker.Currently,tumor specificity exosomes mi RNA(exosomal mi RNA)have been detected in a lot of solid tumors.However,the expression of AML patients' plasma exosomal mi RNA has not been reported.Thus,discussing the difference of AML patients' plasma exosomal mi RNA expression profile and new exosomal mi RNA sequence,and screening out the exosomal mi RNA molecular marker with AML specificity and having an preliminary understanding of its function are of very important and profound significance to explain the molecular mechanism of AML occurrence and development,research and develop new noninvasive diagnostic methods,new diagnostic markers and effectively AML treatment methods.Methods:1?Use total Exosome Isolation Kit and separate exosome from the plasma of 23 cases of early AML patients(AML group)and 23 cases of normal people(control group).2 ?Observe the shape size of exosome through TEM(transmission electron microscopy);and use ZETASIZER Nano series-Nano-ZS instrument to analyze the exosome particle size;use FCM to detect the expression of exosome surface protein CD 63 and CD 81 so as to verify exosome.3?Detect the plasma exosome mi RNA in AML group and control group through high-throughput sequencing,to know the expression of the given mi RNA in samples of two groups.4?Use Mireap prediction software for new miRNAs analysis.5?Normalize the expression quantity of mi RNA through RPM formula into the same order of magnitude so as to analyze mi RNA differential expression in samples of two groups.6?Select mi R-155-5p,mi R-335-5p,mi R-451 a and xxx-m0038-5p(new mi RNA)from mi RNA differential expression in samples of two groups by combining with literatures,and verify plasma exosome samples in two groups(23 cases in each group)through real-time fluorescence PCR(q RT-PCR).7?Prediction of target gene and its function analysis: use mi RNA target gene prediction software to predict the target gen of mi RNA differential expression,and then make GO(Gene Ontology)analysis on predicted target gene and KEGG(Kyoto encyclopedia of genes and genomes)bioinformatics analysis,to have a preliminary understanding of the major biological function of target gene and its participative signal transduction pathway.Results: 1?Exosome separated in this research is in circular or quasi-circular light grey structure of uniform size and uneven distribution;diameter in 30~150nm;the positive expression rate of its surface protein CD 63 is 86.8%,and positive expression rate of CD81 is 92.9%.Both results are positive.2?Successfully construct plasma exosome mi RNA expression profile in AML group and control group through high-throughput sequencing,537 kinds of known mi RNA expression profiles are identified.The expression level of 221 kinds of miRNAs in two groups are obviously different.Compared with control group,expression of 118 kinds of miRNAs are up-regulated and 93 kinds of miRNAs are down-regulated.There are totally 41 kinds of miRNAs with expression quantity difference more than 5 times in two groups.35 kinds of expression quantity are obviously up-regulated in AML group and 6 kinds of expression quantity are obviously down-regulated in AML group.3?Through Mireapprediction software,it is possible that 35 sequences of new mi RNA can be predicted in AML group,221 sequences of new mi RNA can be predicted in control group.Only 7 are expressed in both groups,and only 2(xxx-m0038-5p and xxx-m0022-3p)have obvious differences.4 ? Through real-time fluorescence quantification(PCR)verification,mi R-155-5p,mi R-335-5p,mi R-451 aand xxx-m0038-5p(new mi RNA)have obvious expression differences in the plasma exosome samples in two groups,both 10 times higher than control group,in accordance with sequencing result which is proved to be accurate and reliable.5?Use three software,Target Scan,mi RDB,and mi Randa,and high-flux CLIP-seq database to make target prediction on the first 10 miRNAs with the most obvious expression differences,and obtain 1596 target genes totally.Make GO and KEGG analysis on these target genes,and find that these target genes are obviously enriched mainly in RNA synthetic process regulation,transcriptional control and DNA-template,cell biosynthesis process regulation,gene expression regulation and other GO clauses,and identify17 obviously enriched KEGG signal pathways including Fox O,MAPK,Hippo signal pathway and HTLV-I infection.Conclusions: 1?There were significant differences in the plasma levels of exosomal mi RNA between AML and healthy controls.This research has totally screened out 211 kinds of exosome miRNAs with obviously different expressions.There are totally 41 kinds of miRNAs with expression quantity difference more than 5 times in two groups,which has provided a basis for studies of exosome miRNAs in AML pathogenesis,R & D(research and development)of new noninvasive diagnostic methods and new diagnostic markers.2?Exosome mi R-155-5p,mi R-335-5p,mi R-451 a are very likely to be the molecular markers for AML early diagnosis.3?Discover new mi RNA——xxx-m0038-5p,which may be an important mi RNA related with AML.4?Most biological functions gathered in the target gene of AML plasma exosome mi RNA have participated in biological regulation and relevant pathways of cancer formation.