miR-34a inhibits differentiation of human adipose tissue-derived stem cells by regulating cell cycle and senescence induction

Highlights

• miR-34a expression is increased as the cell passage number is increased in ADSCs.

• miR-34a controls the cell cycle proliferation of ADSCs by regulating cell cycle regulators.

• miR-34a reduces the stemness and differentiation potency of ADSCs.

• miR-34a induces the senescence-inducing IL-6 and −8 production.

Abstract

MicroRNAs (miRNAs) are critical in the maintenance, differentiation, and lineage commitment of stem cells. Stem cells have the unique property to differentiate into tissue-specific cell types (lineage commitment) during cell division (self-renewal). In this study, we investigated whether miR-34a, a cell cycle-regulating microRNA, could control the stem cell properties of adipose tissue-derived stem cells (ADSCs). First, we found that the expression level of miR-34a was increased as the cell passage number was increased. This finding, however, was inversely correlated with our finding that the overexpression of miR-34a induced the decrease of cell proliferation. In addition, miR-34a overexpression decreased the expression of various cell cycle regulators such as CDKs (−2, −4, −6) and cyclins (–E, –D), but not p21 and p53. The cell cycle analysis showed accumulation of dividing cells at S phase by miR-34a, which was reversible by co-treatment with anti-miR-34a. The potential of adipogenesis and osteogenesis of ADSCs was also decreased by miR-34a overexpression, which was recovered by co-treatment with anti-miR-34a. The surface expression of stem cell markers including CD44 was also down-regulated by miR-34a overexpression as similar to that elicited by cell cycle inhibitors. miR-34a also caused a significant decrease in mRNA expression of stem cell transcription factors as well as STAT-3 expression and phosphorylation. Cytokine profiling revealed that miR-34a significantly modulated IL-6 and -8 production, which was strongly related to cellular senescence. These data suggest the importance of miR-34a for the fate of ADSCs toward senescence rather than differentiation.

Introduction

Stem cells are undifferentiated cells that have capacity to self-renew and to differentiate into multiple lineages. They are largely divided into embryonic stem cells (ESCs) and adult stem cells (ASCs) (Passier and Mummery, 2003). ESCs have the greatest differentiation capability, but have considerable challenges for clinical application, including ethical limitations (Borge and Evers, 2003). ASCs are primitive cells that are present in various organs of the adult human body, including the bone marrow, liver, blood, skin and adipose tissues. Although less potent than ESCs, ASCs are more readily available than ESCs, and are multi-potent in that they can differentiate into various lineage cells including neural, osteo-, chondro-, and liver cells. Importantly, ASCs are relatively free from ethical issues because they do not require the use of eggs or embryos, and they have a fewer immunogenic concerns due to their autologous nature (Bunnell et al., 2008). Therefore, ASCs are growing in demand as an alternative source for the use in clinical applications of plastic surgery and regenerative medicine.

Adipose tissue-derived stem cells (ADSCs) are receiving considerable attention because they can be obtained from various parts of the body in relatively high yield, are relatively easy to isolate without much pain, and maintain the typical phenotype of adult stem cells (Bunnell et al., 2008). ADSCs have high proliferation potential, survive well even after long term culture, and show multi-potency with proper induction media (Helder et al., 2007). Although the attempts to apply ADSCs in the medical field are growing, basic research concerning their biological properties require additional characterization. For the safe clinical application of ADSCs, it is essential that we understand more about their biological properties.

MicroRNAs (miRNAs) are short (18–25 nucleotides), endogenous, noncoding RNAs that post-transcriptionally regulate gene expression (Foshay and Gallicano, 2007). Emerging evidence indicates that miRNAs play a critical role in the maintenance, differentiation, and lineage commitment of stem cells in various cellular and biological processes (Foshay and Gallicano, 2007). Importantly, regulation of cell cycle and stem cell transcription factors by miRNAs governs the differentiation potential and specific lineage of stem cells (Foshay and Gallicano, 2007, Carleton et al., 2007, Guo et al., 2011, Marson et al., 2008). Therefore, to understand more about stem cells, we need to better describe and identify biological roles of miRNAs in stem cells.

As stem cells asymmetrically divide resulting in self-renewal or differentiation into cells with specific functions (lineage commitment), determinants of cell specialization may be associated with the cell cycle mechanisms that control proliferation of stem cells (Becker et al., 2006, Fujita et al., 2007). Previously, we have demonstrated the critical role of the cell cycle regulators in the maintenance of the differentiation potential and immaturity of ADSCs (Park et al., 2011). In this study, we have hypothesized that cell cycle-regulating miRNAs might control the differentiation potency of ADSCs. Among the miRNAs that target and regulate cell cycle regulators, we have investigated the role of miR-34a in the differentiation of ADSCs.