Abstract
The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up- and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNA-mediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC.
Highlights
The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive
Raw data filtering of 17,572 analysed transcripts resulted in 12,828 quantified transcripts from which 1,347 mRNAs were significantly upregulated and 800 mRNAs downregulated independent from the time point of XXL-incubation (Supplementary Tables S1 and S2)
Cluster-specific GO-term enrichment analysis indicated that upregulated transcripts in XXL-unrestricted somatic stem cells (USSC) were mainly associated with metabolic processes and negative regulation of cell proliferation whereas downregulated transcripts were involved in cell cycle regulation, DNA replication and repair (Fig. 1A, Supplementary Table S3)
Summary
The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. We evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. As a most important feature, miRNA-mediated regulation is characterised by a bidirectional target gene redundancy This allows a single miRNA to target the 3′ UTRs of hundreds of mRNAs in parallel[2]. Vice versa, binding sites for several miRNAs can be found on a single 3′ UTR In consequence of these properties, miRNAs primarily can act as network regulators being able to affect large numbers of regulatory targets in parallel[2,3]. Since USSC treated with XXL for 14 days lack action potentials they must be considered as only partially differentiated cells
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