Quantitative profiling of differentially expressed miRNAs in exosomes of siCTL- and siPRDX3-transfected HCT 116 cells. Quantitative profiling of differentially expressed miRNAs in exosomes of siCTL- and siPRDX3-transfected HCT 116 cells

Purpose: The goal of this study is to compare exosomal small RNA transcriptome of HCT116 cells to identify the target of PRDX3 under basal or knock down conditions by utilizing miRNA-seq. Methods: miRNA profilies of siCTL or siPRDX3 transfected HCT 116 exosoems were generated by illumina sequencing method, in triplicate. After sequencing, the raw sequence reads are filtered based on quality.Sequence reads were mapped with the bowtie2 software tool, which yielded bam files. Mature miRNA sequences were used as references for mapping. Read counts mapped to a mature miRNA sequence were extracted from the alignment file using bedtools v2.25.0 and Bioconductor, which use the R statistical programming language. Read counts were used to determine the expression level of miRNAs. The CPM+TMM normalization method was used for between-sample comparison. Results: We identified known miRNA in species (miRDeep2) in the HCT116 exosome transfected with siCTL or siPRDX3. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of HCT116 exosomal miRNA profiles affected by PRDX3 knockdown with biologic replicates. Overall design: The HCT-116 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 saturated humidity. The PRDX3 siRNA, control siRNA were transfected using a NEON microporation system for 40hours.To isolate exosomes, cells were placed in RPMI 1640 with 10% nanoparticle-free FBS and incubated for 24 h, and exosomes were isolated using an ExoLutE exosome isolation kit according to the manufacturer’s instructions. Endothelial micro-RNA profiles of siCTL- and siOASL-transfected HUVECs were generated by miRNA-seq.