Role of MicroRNAs in LPA-Induced Regulation of Stem Cell Differentiation Into Cardiomyocytes

Abstract

Lysophosphatidic acid (LPA) is known to exert a diverse range of effects in humans, specifically in the heart where it may cause apoptosis of cardiomyocytes at pathological concentrations. However, at physiological concentrations LPA may regulate cell migration, proliferation and even differentiation. The ability to drive stem cells down the cardiac lineage has, however not yet investigated and the potential underlying molecular mechanisms that could mediate this effect also remains to be determined. Since LPA is reported to accumulate in acute myocardial infarction and may rescue cardiac myocytes, we have hypothesised it may also be able to generate cardiac myocytes from stem cells. This hypothesis is based on observations that the signalling through which it exerts its biological effects are similar to those which have reported regulating stem cells regulation to various lineages. The aim of this thesis, therefore, was to study, firstly, the effectiveness of LPA in deriving cardiomyocytes and then to establish the molecular mechanisms involved focusing specifically on the expression profile of select miRNAs including mir-145, mir-1 and mir-133 which respectively linked to pluripotency and lineage commitments. All studies were carried out using the P19 stem cell line and were maintained and cultured in supplemented alpha-minimal essential medium (α-MEM), comprised of antibiotics and foetal bovine serum (FBS). Embryoid bodies were formed from aggregates of the cells in a non-tissue culture grade plates with or without LPA for four days. These EBs are subsequently plated in an adherent 6-well plate and maintained in culture between 3 and 12 days, before being lysed for either western blotting or RNA analysis. When used pharmacological inhibitors of LPA receptors (Suramin (P2 purinergic/LPA receptor 4), H2L5765834 (LPA receptor 1, 3 & 5), H2L5186303 (LPA receptor 2 & 3) and TC-LPA5-4 (LPA receptor 5)), targeted protein kinases (PKC; by BIM I) and PI3-kinase (by LY294002), they were added to the cultures 1 hour before treatment with LPA. In parallel studies, cells were transfected with mir-145 and mir-1 using siRNAs inhibitors or overexpression. The effect on MLC-1v and OCT4 protein expression then determined by western blotting. Successfully, the research revealed that LPA could achieve differentiation of P19 stem cells, and this was concentration and time-dependent. The maximum response was obtained with 20μM LPA and peaked on day 6. Subsequent experiments were carried out using 5 and/or 20μM LPA. Decreases paralleled the induction of MLC-1v in OCT4 expressions. The effects of LPA were mediated through its receptors, specifically LPA receptors 4 and 5 and partially on LPA receptors 2 and 3. The effects were also mediated through the kinases like protein kinase C, although the involvements of PI3 kinase was only partial. Expressions of mir-145, mir-1 and mir-133 elevated following treatment with LPA. Suramin, BIM-I inhibited these changes but not affected by LY294002, H2L5186303, H2L5765834 and TC-LPA5-4. Transfection studies also suggested that mir-1 regulate differentiation through enhanced MLC-1v protein expression with and without LPA. In conclusion, the program of research conducted for this thesis has confirmed that LPA could be an endogenous modulator of stem cells differentiation to a cardiac lineage, more importantly this effect is mediated through its receptors 2, 3, 4 and 5 collectively, that may activate protein kinase PKC and PI3K and is under the influence of mir-145 and mir-1. Our work in this thesis suggests the active role of miRNAs specifically mir-145 and mir-1, which can indicate the need of further investigation to instigate the possible therapeutic targets and further prevent any fatal consequences of atherosclerosis or other heart-linked diseases.