Functional omics analyses reveal only minor effects of microRNAs on human somatic stem cell differentiation

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 miRNAmediated 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. MicroRNAs (miRNAs) are small non-coding RNAs which primarily bind to the 3′UTR of target mRNAs in a sequence-specific manner resulting in translational repression or destabilization and degradation of the targeted mRNA 1. In animals, primary miRNA transcripts are trimmed in a two-step process involving double strand-specific nucleases Drosha and Dicer. The mature single-stranded miRNA is then incorporated into the RNA-induced silencing complex (RISC) where functional binding to the mRNA target sequence takes place 1. 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 .

Transcription factor RFX7 governs a tumor suppressor network in response to p53 and stress

“…Complementary data displayed in Supplementary Figure 3. (C) Gene set enrichment analysis (GSEA) of 57 direct RFX7 target genes in human p53-negative HL-60 promyelocytes differentiated into macrophages or neutrophils (upper panels) (47), human umbilical cord blood-derived unrestricted somatic stem cells (USSC) differentiated into neuronal-like cells (48) and human pluripotent stem cells (PSC) differentiated into lung alveolar cells (bottom panels) (49).…”

“…Publicly available RNA-seq data from human p53-negative HL-60 promyelocytes differentiating into macrophages or neutrophils was obtained from GEO accession number GSE79044 (47). Publicly available RNA-seq data from of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) differentiating into neuronal-like cells was obtained from GEO accession number GSE96642 (48). Publicly available RNA-seq data from human pluripotent stem cells differentiating into lung alveolar cells was obtained from GEO accession number GSE96642 (49).…”

“…Sequencing data is accessible through GEO (55) series accession numbers GSE162157, GSE162158, GSE162159, GSE162160, GSE162161, GSE162163. Previously published RNA-seq data was obtained from GEO accession numbers GSE113267 (17), GSE79044 (47), GSE144464 (48), and GSE96642 (49). Previously published p53 ChIP-seq data was obtained from CistromeDB (40) IDs 82544 (43) and 33077 (42).…”

“…We reveal that RFX7 orchestrates multiple established tumor suppressor genes in response to cellular stress. Thus, RFX7 emerges as a crucial regulatory arm of the Publicly available RNA-seq data from of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) differentiating into neuronal-like cells was obtained from GEO accession number GSE96642 (48). Publicly available RNA-seq data from human pluripotent stem cells differentiating into lung alveolar cells was obtained from GEO accession number GSE96642 (49).…”

MicroRNA‑489‑3p plays a significant role in congenital hypothyroidism through regulating neuronal cell apoptosis via targeting translationally controlled tumor protein 1

“…MicroRNAs (miRNAs) are highly conserved small RNAs, 20-22 nucleotides in length, which modulate protein function via directly binding to the 3'-untranslated region (3'-UTR) of their target mRNAs ( 11 ). Recent studies have demonstrated that changes in miRNA levels are involved in numerous diseases, including myocardial ischemia ( 12 ), inflammation ( 13 ), diabetes ( 14 ) and CH ( 4 , 8 , 9 ).…”