The role of microRNA-302 in regulation of human bone marrow Mesenchymal Stem Cell fate determination

Student thesis: Doctoral ThesisDoctor of Philosophy


Mesenchymal stem cells (MSCs) are adult stem cells which are characterised by self-renewal and differentiation capacity into three mesenchymal lineages: osteoblast, chondrocyte and adipocytes. MicroRNAs (miRNAs) are a class of non-coding short RNAs (20-22 nucleotides) which target messenger RNAs to supress protein synthesis and regulate cell function. There is increasing evidence that miRNAs can play key roles in the regulation of multiple biological processes include stem cell regulation. Therefore, the aim of this study was to investigate the potential role of miRNAs in the regulation of MSC fate decisions. Firstly, 4 different MSC cell lines were tested for their differentiation capacity following induction with osteogenic, adipogenic, or chondrogenic media. From these results one cell line was selected for use in further studies. The expression of 9 candidate miRNAs which had previously been shown to be associated with regulation of other stem cells were tested during osteogenic or adipogenic induction. To identify novel miRNAs associated with MSC regulation, clonally derived MSCs with osteogenic, adipogenic or bipotent differentiation patterns were tested using miRNA array. From the results of the array 2 family members of the miRNA-302 (a and b) were selected for further investigation. MiR-302a and b expression was validated by qRT-PCR in the clonally derived cell lines and in primary MSC cultures. To determine the role of the miR-302 family during MSC differentiation, their functional activity was tested by knock-down and over expression, via transfection of a miRNA-302 family inhibitor or individual miR-302a and miR-302b mimics into MSCs and the cells were treated with osteogenic, adipogenic or growth medium as control for 14 days. Then lineage specific gene expression was measured by qRT-PCR. Expression patterns of the 9 candidate genes showed no reproducible significant effects during osteoblastic or adipocytic differentiation. MiRNA-302a and b were both down regulated during differentiation of MSCs into both osteogenic and adipogenic lineages. Knock-down of miRNA-302 family in MSCs caused a significant upregulation of between 2-5 times of RUNX2 expression and between 3-10 times of ALP expression during osteogenic differentiation of MSCs. Overexpression of miR-302b did not have any significant reproducible effects on osteogenic differentiation of MSCs. In contrast, over expression of miR-302a resulted in increased RUNX2 expression by 1.5-2 fold on day 2 and ALP expression by almost 50 fold after day 4. Inhibition or over expression of miR-302 family had no early effects on adipogenesis of MSCs, but at day 14 FAB4 was upregulated nearly 40% in inhibited cells and between 20-30% in miR-302a overexpressed cells. In contrast overexpression of miR-302b downregulated FAB4 by a range of between 10-80% at day 14. ALP enzyme activity rose at day 7 after inhibition of miR-302 and decreased at day 10 and 14 after miR-302a over expression significantly. The results suggest that miRNA-302 acts to maintain MSC phenotype by specific repression of osteogenic differentiation. The results also suggest that this may be specifically due to the action of miR-302a. In contrast the miR-302 family had little or no effects on adipogenic differentiation of MSCs, with the exception of a transient upregulation of FAB4 at a late stage of differentiation due to miR-302 inhibition.
Date of Award1 Jun 2019
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorFrancis Hughes (Supervisor) & Mandeep Ghuman (Supervisor)

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