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MiRNA expression profiles of EVs from control and MDS mice

ABSTRACT: To identify the mechanism by which miRNAs in EVs derived from MDS cells suppressed the osteolineage differentiation of MSCs and disrupted normal hematopoiesis, we comprehensively explored the miRNAs encapsulated in EVs by miRNA-array. Consistent with the heterogeneous nature of MDS, the hypoplastic Abcg2-MDS/AML model and the hyperplastic NHD13Tg model share few elevated miRNAs. However, it is noteworthy that the pathways targeted by each upregulated miRNA are mutually shared, such as the pathways associated with MSC differentiation and survival, including axon guidance and MAPK signaling. These results suggest that miRNAs play an essential role in the MSC impairment observed in MDS.

ORGANISM(S): Mus musculus

PROVIDER: GSE199997 | GEO | 2022/10/01

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.

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MiRNA expression profiles of EVs from control and MDS mice

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