Study Of Multi-directional Regulation Of Endothelial Progenitor Cell Differentiation By Micro RNA 126

Background and objectiveEndothelial progenitor cells(EPCs) play an important role in endothelium repair. When the vascular endothelium is injured, circulating EPCs, are mobilized from the bone marrow(BM), migrate to the ischaemic tissue, differentiate to mature vascular endothelial cells, and then to repair the injured endothelium. However, BM-derived EPCs also have the ability to transdifferentiate into a smooth muscle cell lineage positive for alpha smooth muscle actin(α-SMA), i.e., endothelial-tomesenchymal transition(End MT), suggesting a contributive role for EPCs in intimal hyperplasia during the endothelial repair process. In End MT, EPCs lose the expressions of endothelial markers such as KDR, CD34 and VEGF, and begin to express mesenchymal markers such as α-SMA, SM22α and myocardin.microRNA 126(miR-126) is highly expressed in vascular endothelial cells and promotes angiogenesis. Accumulating evidence has demonstrated that mi R-126 could fuel the cellular mobilization, migration and proliferation of EPCs, instead of the differentiation to endothelial cells. However, the role of mi R-126 in regulating the transition of EPCs to mesenchymal cells has not been reported to date.In the present study, we assessed the effects of mi R-126 on TGF-β1-induced End MT of EPCs isolated from the bone marrow(BM). Additionally, we investigated the roles of PIK3R2 and the PI3K/Akt signalling pathway in the End MT process of EPCs regulated by mi R-126.MethodsRat bone marrow EPCs were isolated with Ficoll-Isopaque Plus density-gradient centrifugation. Daily observation for morphology and growth was performed under phase contrast microscopy. The purity of EPCs were determined by using immunofluorescence staining assay, flow cytometry and double immunofluorescence staining for Di I-Ac-LDL and FITC-UEA-1. EPCs End MT were induced by TGF-β1(5 ng/ml in medium) for 7 days. Overexpression of mi R-126 constructed in EPCs by using the transfection with lenti-mi R-126 plasmid. Cellular viability was detected byusing MTT assay. Cellular migration was detected by using cell injury and Transwell assay. EPCs were transfected by PIK3R2 sh RNA lentivirus to knockdown PIK3R2 expression. PIK3R2 c DNA lenti-vector was constructed and transfected into EPCs to induce overexpression of PIK3R2. Luciferase reporter assay was used to identify a potential target gene, PIK3R2, of mi R-126. EPCs were transfected by Fox O3 sh RNA lentivirus to knockdown Fox O3 expression. Mesenchymal transcription factors were detected by using quantitative real-time PCR. Signaling molecules expressions were measured by using western-blotting assay.Results1. EPCs End MT were induced by TGF-β1(5 ng/ml) for other 7 days seccessfully. The expression of mi R-126 of EPCs treated by TGF-β1(5 ng/ml) was downregualted gradually in a time-dependent manner. Lenti-mi R-126 did not affect EPCs aviability, however, inhibited the proliferation and migration of EPCs treated by TGF-β1.2. Overexpression of mi R-126 inhibited the TGF-β1-induced m RNA expression of mesenchymal markers(α-SMA, SM22α, and myocardin) and mesenchymal transcript factors(slug, snail, zeb1, endothelin-1) as well as α-SMA protein in EPCs. In addition, mi R-126 maintain m RNA expressions of progenitor cell markers(CD34, CD31, CD133, and KDR) and endothelial cell markers(VEGF, Flt-1, e NOS, and i NOS).3. PIK3R2 was demonstrated as the target gene of mi R-126 in EPCs by using luciferase reporter assay. PIK3R2 sh RNA induced a morphological change in EPCs from a spindle shaped to angular appearance, and an increase of α-SMA and SM22α protein expressions.4. Mi R-126 decreased the protein level of PIK3R2 and SM22α in EPCs treated by TGF-β1, and such an effect of mi R-126 was reversed by PIK3R2 c DNA.5. There were a decrease of PI3 K, p-Akt and p-FoxO3, and an increase of p-Smad3 in EPCs induced by TGF-β1. These TGF-β1-induced changes were reversed by PIK3R2 sh RNA. And the effects of PIK3R2 sh RNA were reversed by the pretreatment with PI3 k inhibitor LY294002. There was no change of Smad3, p-Smad and Smad4 in any group.6. IGF-1, an activator for PI3K/Akt, increased expressions of PI3 K, p-Aktaccompanied by an increase of p-Fox O3, and decreased expressions of SM22α in EPCs induced by TGF-β1. but had no effect on the expression of Smad3, p-Smad and Smad 4.7. Compared to control EPCs, there were a decrease of PI3 K, p-Akt and p-Fox O3 in EPCs induced by TGF-β1. These TGF-β1-induced changes were reversed by mi R-126. Such an effect of mi R-126 in EPCs was reversed markedly by PIK3R2 c DNA. There was no change of p-Smad3 and Smad4 in any group. TGF-β1 induced an increase of nuclear expressions of Smad4 and Fox O3 in EPCs, which was reversed by mi R-126 markedly.8. Compared to control EPCs, TGF-β1 induced an increase of SM22α, and a decrease of p-Fox O3. These TGF-β1-induced changes were reversed by Fox O3 sh RNA.ConclusionsThe EPCs EndMT was associated with PI3K/Akt-FoxO3 signaling pathway. Overexpression of mi R-126 could inhibit EPCs End MT. Mi R-126 inhibits EPCs End MT via targeting the gene PIK3R2, increasing phosphorylated level of PI3K/Akt, promoting the phosphorylation of Fox O3 and inhibiting nuclear translocation of Smad 4 consequently induced by TGF-β1.