Effect of BMSCs-derived exosomes on expression of lncRNAs in rat carotid artery after balloon injury

Abstract

Objective To investigate the regulatory effect of bone marrow mesenchymal stem cells (BMSCs) derived exosomes on intimal hyperplasia and expression of long non-coding RNAs (lncRNAs) in rat model of carotid artery balloon injury and its potential mechanism. Methods Rat BMSCs-derived exosomes were extracted from male SD rats by differential centrifugation, and then identified by Western blotting and transmission electron microscopy. Then a total of 36 male SD rats were randomly divided into sham-operation group, model group (carotid artery balloon injury), and model+exosome treatment group (in situ injection of 400 μL 0.25 μg/μL exosomes), with 12 rats in each group. In 14 d after the treatment, all rats were sacrificed to harvest the carotid artery. HE staining was used to observe the intima/media (I/M) area ratio of carotid artery. Immunohistochemical assay was used to detect the expression of PCNA, and Western blotting was applied for the expression of the proteins involved in the intimal proliferation and phenotypic transformation. LncRNA-mRNA co-expression microarray and bioinformatics analysis were employed to analyze the differentially expressed lncRNAs and mRNAs in carotid artery tissues of the rats from the sham operation group and the model group for the potential molecular mechanism. qRT-PCR was performed to detect the expression of candidate lncRNAs in the carotid artery tissues. Results Transmission electron microscopy displayed that the BMSCs-derived exosomes presented "coaster-like" changes. Western blotting results confirmed that CD63, Alix and HSP70 were expressed in the extracted exosomes. The treatment of BMSCs-derived exosomes resulted in significant reduce in the I/M area ratio of carotid artery (P 2.0, P < 0.05) in the model group when compared with the sham operation group. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these multiple lncRNAs were correlated with mRNAs, and the corresponding target mRNAs are involved in many biological processes, such as vascular smooth muscle contraction, actin cytoskeleton regulation and cell cycle, which are related to the mechanism of vascular restenosis after balloon injury. In combination with bioinformatics analysis, qRT-PCR detected 7 lncRNAs with significant differential expression, and the results showed that XR_593268 was up-regulated (P < 0.05), and XR_602080, XR_602012 and XR_591017 were down-regulated (P < 0.05) in the exosome treatment group compared with the model group. Conclusion The BMSCs-derived exosomes inhibit intimal hyperplasia in rats after balloon injury of carotid artery. The potential mechanism may be related to the regulation of smooth muscle cell cycle changes by lncRNAs XR_602080 and XR_591017 through Gsk3b, Cdkn2b and Ccna2.

Keywords

• bone marrow mesenchymal stem cells
• exosomes
• carotid artery injury
• long non-coding rna