Up-regulation of Liver Enriched Transcription Factors (HNF4a and HNF6) and Liver Specific MicroRNA (MiR-122) by Inhibition of Let-7b in Mesenchymal Stem Cells

Nosratollah Zarghami1,*, Effat Alizadeh2, Abolfazl Akbarzadeh3, Mohamadreza Baghaban Eslaminejad4, Kazem Nejati-Koshki5

1Department of Clinical Biochemistry and Medical Biotechnology, Faculty of Medicine, 2Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, 3Department of Medical Nanotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz University of Medical Sciences, Tabriz, 4Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 5Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, Islamic Republic Of

Introduction: MicroRNAs are small non-coding RNAs that regulate key processes of the stem cells. Although, microRNAs have emerged as powerful regulators of differentiation; few studies have been focused on the post transcriptional regulation of hepatic differentiation in mesenchymal stem cells (MSCs) by microRNAs.

Objectives: The aim of the present study was to evaluate the specific effect of let-7 microRNAs in particular let-7b in hepatic commitment of human adipose tissue derived mesenchymal stem cells (hAT-MSCs).

Methods: After isolation of hAT-MSCs from adipose tissue, the identity and multi-potency of them were confirmed. Hepatic differentiation of hAT-MSCs was induced and functionality of hepatocyte like cells (HLCs) derived from hAT-MSCs was investigated. The dynamic expression profile of let-7a, b, c microRNAs, and two liver enriched transcription factors (LETFs) HNF4a and HNF6 were investigated during in vitro hepatic differentiation of hAT-MSCs by LNA-based qRT-PCR. HAT-MSCs were transfected with let-7b mimic, inhibitor, and negative control using lipofectamine 2000, subsequently, the alterations of HMGA2, let-7b, Lin28, HNF4a, HNF6, miR-122, and cell cycle were surveyed

Results: It was shown that the expression of LETFs is inversely correlated with those of let-7 miRNAs during differentiation progress (p<o.05).Upon over expression of let-7b, down-regulation of LETFs was observed. Furthermore, inhibition of let-7b caused an increase in the expression levels of liver specific miRNA, miR-122(3 fold, p<0.01) emulating the features of functional hepatocytes and accumulation of hAT-MSCs in the G0/G1phase of cell cycle, triggering initiation of hepatic commitment.

Conclusion: Transient inhibition of let-7b activates in vitro hepatic differentiation of hAT-MSCs. The findings of this work might help optimization of an invitro hepatogenic differentiation utilizing microRNAs and hAT-MSCs could be used for therapeutic purposes.


Keywords: Liver, Other organ, RNA