Improved therapeutic effects on vascular intimal hyperplasia by mesenchymal stem cells expressing MIR155HG that function as a ceRNA for microRNA-205

Abstract

Coronary artery bypass grafting (CABG) is an effective treatment for coronary heart disease, with vascular transplantation as the key procedure. Intimal hyperplasia (IH) gradually leads to vascular stenosis, seriously affecting the curative effect of CABG. Mesenchymal stem cells (MSCs) were used to alleviate IH, but the effect was not satisfactory. This work aimed to investigate whether lncRNA MIR155HG could improve the efficacy of MSCs in the treatment of IH and to elucidate the role of the competing endogenous RNA (ceRNA). The effect of MIR155HG on MSCs function was investigated, while the proteins involved were assessed. IH was detected by HE and Van Gieson staining. miRNAs as the target of lncRNA were selected by bioinformatics analysis. qRT-PCR and dual-luciferase reporter assay were performed to verify the binding sites of lncRNA-miRNA. The apoptosis, Elisa and tube formation assay revealed the effect of ceRNA on the endothelial protection of MIR155HG-MSCs. We observed that MIR155HG improved the effect of MSCs on IH by promoting viability and migration. MIR155HG worked as a sponge for miR-205. MIR155HG/miR-205 significantly improved the function of MSCs, avoiding apoptosis and inducing angiogenesis. The improved therapeutic effects of MSCs on IH might be due to the ceRNA role of MIR155HG/miR-205.

Keywords: MIR155HG; ceRNA; intimal hyperplasia; mesenchymal stem cells; microRNA‐205.

FIGURE 1

MSCs and MIR155HG transfection. (A) Microscopic view of fibroblast‐like MSCs. (B) CXCR4 (red) and DAPI (purple) immunostaining on MSCs. (C) Surface makers of MSCs by flow cytometric analysis. Cells were incubated with CD29, CD34, CD45 and CD90 antibodies. (D) Green fluorescence of MSCs transfected with lentivirus observed under fluorescence microscope. (E) Transfection efficiency of MIR155HG in MSCs verified by qRT‐PCR, *p < 0.05. (F) The expression of RAP1 in MSCs, *p < 0.05.

FIGURE 2

Effects of MIR155HG and NF‐κB pathway on proliferation and migration of MSCs. (A) Representative images of flow cytometry. (B) Cell distribution in different phase of the cell cycles determined by flow cytometry, *p < 0.05 or ^† p < 0.05 vs. MIR155HG group. (C) Representative images of transwell assay. (D) Quantitative analysis of cell migration, *p < 0.05 vs. MIR155HG group. (E) Images of the transwell chambers used in the experiment. (F) Representative western blots of p‐NF‐κB p65/ NF‐κB p65 expression in MSCs. (G) Quantitative analysis of western blots, *p < 0.05 vs. MIR155HG group.

FIGURE 3

Role of MIR155HG in the treatment of IH by MSCs. (A) Representative HE staining images (×15) of vein graft. The black arrow indicates the intima of the vein graft. (B) Representative Van Gieson staining images (×30) of vein graft. The collagen is stained red and the muscle is stained yellow. (C, D) Quantitative analysis of A and B, *p < 0.05. (E) Rat model of autogenous vein transplantation. The white arrow indicates the grafted vein, the black arrow indicates the abdominal aorta.

FIGURE 4

Sequencing analysis of differentially expressed MIR155HG‐related miRNAs. (A) Venn diagram of predicted results of the targeted genetic locus. (B) Clustering heat map of differentially expressed miRNA. The clustering of miRNAs with similar expression patterns into classes was used to predict the functions of unknown miRNAs or new functions of known miRNAs. (C) Volcano maps of differentially expressed miRNA. The volcano map was used to evaluate the differences in miRNA expression in the two different samples, as well as the statistical significance of the differences. (D) Differentially expressed miRNA. (E) GO functional classification map of differentially expressed miRNA.

FIGURE 5

MIR155HG functions as a ceRNA for miR‐205. (A) Clustering heat map of the top 10 significantly up‐regulated and down‐regulated miRNAs. (B) Results of the combined analysis of miRNAs and MIR155HG by miRanda software according to threshold values. (C) Specific binding sequences of MIR155HG to miR‐205. (D) Dual‐luciferase reporter assay was used to detect the binding sites between MIR155HG and miR‐205, *p < 0.05; ^# p > 0.05.

FIGURE 6

miR‐205 suppresses the viability and migration of MSCs. (A) Reverse transcription and primer sequence of miR‐205. (B) Transfection efficiency of miR‐205 in MSCs by qRT‐PCR, *p < 0.05. (C) Cell viability by CCK‐8 assay, *p < 0.05. (D) Representative images of transwell assay. (E) Migratory ability of MSCs after miR‐205 transfection, *p < 0.05. (F) Representative western blots of p‐NF‐κB p65 expression in MSCs. (G) Quantitative analysis of western blots, *p < 0.05.

FIGURE 7

MIR155HG improves apoptosis and angiogenesis in vitro by sponging miR‐205. (A) Representative images of cell apoptosis detected by flow cytometry. (B) Analysis of the role of miR155HG/miR‐205 ceRNA on the apoptosis of MSCs, *p < 0.05. (C) Representative images of endothelial cell tube formation. The indicators of tube formation were analysed by Image J software. (D, E) Quantitative analysis of tubular forming ability of HUVECs, *p < 0.05. (F) Secretion of VEGF in the supernatant of MSCs detected by Elisa, *p < 0.05.