Title: Differential MicroRNA Expression Profiling In Vascular Endothelial Differentiation Of Adipose Derived Stromal/Stem Cells

Objectives: The key point of this research was vascular endothelial differentiation ofADSCs. We applied the miRNA microarray assay and qRT-PCR to the test for thedifferentially expressed miRNAs during the differentiation, and then further predicted thetarget genes of these miRNAs and analyzed in which signaling pathway they probablyparticipated.Methods:1Cultured and passaged hADSC, then induced its differentiation into vascularendothelial cells.2Phenotyped hADSC and the differentiated vascular endothelial cells by flowcytometry. Observed the histology of them by optical and electron microscope. All theseprocedure aimed to guarantee the reliability of the results.3Extracted the total miRNAs from ADSC and vascular endothelial cells, and thenanalyzed the differentially expressed miRNAs between these2groups of cells by miRNAmicroarray assay.4According to the result of the miRNA microarray assay, selected the miRNAswith the higher fold change, used qPT-PCR to confirm the differential expression of themfurther.5Analyzed the Pathway and GO of the miRNAs that were obtained from themiRNA microarray assay by bio-informatics software David6.76Searched the online databases: TargetScan，Miranda，PITA，RNAhybrid，microTar;to predict the target genes of the miRNA obtained from the qRT-PCR, the target genesexisting in more than (including)3databases were selected to do the further analysis.Results：1Phenotyping by flow cytometry： hADSC was positive for CD44and CD105; negative for CD31and HLA-DRthe differentiated vascular endothelial cells was positive for CD31and CD105;negative for CD44and HLA-DR.2Optical and electron microscope analysis:Optical: the shape of the hADSC is long, arranged whirly; the vascular endothelialcells were round;Electron: W-P body existed in the cytoplasm of the vascular endothelial cells, not inthe hADSC.3miRNA microarray assay and qRT-PCR analysis:miRNA microarray assay: when P value〈0.05, there’re30differentially expressedmiRNAs existed during the differentiation of hASDC,16were up-regulated after thedifferentiation,14were down-regulated.qRT-PCR: from the6miRNAs selected from the miRNA microarray assay, whichhad higher fold change of differential expression，4had significant differential expressiondue to tqRT-PCR, which were up-regulated: has-miR-29b-3p， has-miR-5096；down-regulated: has-miR-143-3p，has-miR145-5p。4Target genes prediction of the differentially expressed miRNAs and pathwayanalysis of these target genesThe signaling pathways in which these target genes participated involve in the samepathway of tumorgenesis.Conclusions:1There exist differentially expressed miRNAs during the angiogenesis of ADSC,including up-regulated and down-regulated expression.2The signaling pathways in which the target genes of these differentially expressedmiRNAs participate, also involve in the same pathway of tumorgenesis.