Prognostic Correlation Analysis And Clinical Verification Of AML Mediated By MiR-20b And MiR-363

Objective: TCGA database was used to mine miRNA associated with poor prognosis of AML,and then explored the expression and clinical significance of differential miRNA in AML patients with poor prognosis.Method: AML related mi RNA-Seq data and clinical data were downloaded from the TCGA database,then divided the clinical data into three groups: favorable,inter mediate and adverse.Differential expression miRNA were screened between favorable group and adverse group.RT-qPCR was used to detect the expression of miR-20b-5p and miR-363-3p in AML patients.Survival analysis of differential miRNA(miR-20 b,miR-363)and ROC curves were performed based on the data from the TCGA.The miRNA expression levels were divided into low expression group and high expression group,then analyzed the correlations in clinicopathological parameters(age,sex,white blood cell number,FLT3 mutation and NPM1 mutation).Find the mature sequences of differential expressed miRNA in multiple specie,then analyzed the conservatism in different species.The target genes of differential expressed miRNA were predicted by four online software: Targetscan,miRanda,miRDB and DIANA-microT,took the sum aggregate of the target genes which were predicted by four kinds of software and the target genes that have been supported by experimental data in miRTarBase and miRPathDB databases as a set of target genes for subsequent analysis.GO annotation and KEGG enrichment analysis of target gene set were carried out by DAVID online database.Plotted the protein interaction network by STRING online analysis software,and the protein interactions were graphically displayed in the Cytoscape software,the plug-in Cytohubba was used to select hub genes.Result: 1.By analyzing the data of AML related miRNA expression in TCGA database,54 miRNAs with obvious differential expression were obtained,of which 49 were up-regulated and 5 were down-regulated.RT-qPCR results showed that the expression levels of miR-20b-5p and miR-363-3p in adverse group of AML were significantly higher than those in favorable group(P<0.05).2.The TCGA data survival analysis showed that miR-20 b and miR-363 were associated with the prognosis of AML patients,and the survival of the patients with miRNA high expressed was shortened.3.There wasa statistical difference between miR-20 b,miR-363 and age,white blood cell count of AML patients(P<0.05),but there was no significant difference in sex,FLT3 and NPM1group(P>0.05).4.MiR-20b-5p and miR-363-3p mature sequences were highly similar among 12 species.158 miR-20b-5p target genes and 282 miR-363-3p target genes were obtained through online prediction database.The results of GO and KEGG analysis showed that miR-20b-5p target genes were mainly enriched in biological processes and molecular functions such as transcription,protein phosphorylation,cell cycle and cell apoptosis,mainly involved in the cancer pathway,ErbB,HIF-1,Fox O and PI3K/Akt signaling pathway.The miR-363-3p target genes were mainly enriched in biological processes and molecular functions such as transcription,translation,ubiquitination,cell cycle,cell proliferation and cell apoptosis,they were mainly involved in cancer pathways,P53,FoxO,PI3K/Akt signaling pathways and platelet activation(P<0.05).5.The top hub genes of miR-20b-5p and miR-363-3p selected by Cytoscape software were CDKN1 A,NEDD4L,ITCH,HIF1 A,HECTD2 and PIKFYVE,PHLPP2,PTEN,ACTC1,RAP1 B.Conclusion: 1.A total of 54 differentially expressed miRNA were successfully screened in the AML poor prognosis group.MiR-20 b and miR-363 were up-regulated among them.There was a significant positive correlation between the two mi RNA.2.The survival time of patients with high expression of miR-20 b and miR-363 was shortened,which could be used as an auxiliary diagnosis index for AML prognostic stratification.3.The expression levels of miR-20 b and miR-363 were significantly correlated with age and white blood cell counts,but not related to sex,FLT3 and NPM1.4.MiR-20b-5p and miR-363-3p target genes were involved in multiple biological processes and signal transduction pathways by regulating the expression of a series of target genes.5.Successfully constructed PPI network of target genes and screened the hub gene.