RESUMO
AIM: MicroRNAs (miRNAs) constitute a class of small non-coding RNAs involved in regulation of cognate mRNAs post-transcriptionally. MicroRNAs have been implicated in regulating the stem cell differentiation process. Limited regulatory miRNAs have been reported to date during hepatic differentiation of stem cells. The present study was designed to identify the signature miRNAs implicated in hepatic differentiation of stem cells using next-generation sequencing methods. METHODS: We undertook sequencing of miRNAs isolated from three different time points during hepatic differentiation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hUC-MSCs) from two biological replicates. RESULTS: Out of a total known 2588 miRNAs (according to miRBase version 21), 880 miRNAs were identified in our study. A total of 63 significantly expressed miRNAs during hepatic differentiation, with at least 2-fold change and a false discovery rate value <0.05, were considered for further analysis. The putative target genes of significantly downregulated miRNAs during hepatic differentiation appeared to be mostly associated with biological processes that are essential for hepatic differentiation and maintenance of mature hepatic phenotype-like liver development, stem cell differentiation, Wnt receptor signaling pathway, and drug and cholesterol metabolic processes. Putative target genes of significantly upregulated miRNAs are highly enriched in regulating processes that block hepatic differentiation of hUC-MSCs like epithelial-mesenchymal transition, transforming growth factor-ß receptor signaling pathway, and stem cell maintenance. CONCLUSION: The study provides a new insight for investigation of miRNA-regulated pathways during the differentiation process.
RESUMO
MicroRNAs (miRNAs) play an important role in the control of cell fate determination during differentiation. In this study, we analyzed the expression pattern of microRNAs (miRNAs) during hepatic trans-differentiation. The protocol employed the use of histone deacetylase inhibitor (HDACI), valproic acid (VPA) to induce hepatic trans-differentiation of human umbilical cord Wharton's jelly derived mesenchymal stem cells (hUC-MSCs). The differentiated hepatocyte like cells (HLCs) from hUC-MSCs shared typical characteristics with mature hepatocytes, including morphology, expression of hepatocyte -specific genes at the molecular and cellular level. Moreover, the functionality of HLCs was confirmed through various liver function tests such as periodic acid-Schiff (PAS) stain for glycogen accumulation, enzyme-linked immunosorbent assay (ELISA) for synthesis of albumin and release of urea. The aim of the present work was to examine the effect of VPA treatment on miRNA expression during hepatic trans-differentiation. The analysis at miRNA level showed that there was a significant increase in expression of miRNAs involved in hepatic differentiation, due to VPA pre-treatment during differentiation. The study, thus demonstrated that improved expression of hepatocyte-specific miRNAs, miR-23b cluster (miR-27b-3p, miR-24-1-5p and miR-23b-3p), miR-30a-5p, miR-26a-5p, miR-148a-3p, miR-192-5p, miR-122-5p due to VPA pre-treatment contributed to a more efficient hepatic trans-differentiation from hUC-MSCs. The putative targets of these upregulated miRNAs were predicted using Bioinformatics analysis. Finally, miR-122-5p, highly upregulated miRNA during hepatic differentiation, was selected for target verification studies. Thus, this study also provides the basis for the function of miR-122-5p during hepatic differentiation of hUC-MSCs.
