Inhibition of miRNA-21-5p Rescued the Disrupted Tight Junction and Promoted Apoptosis in CCM1-Deficient Endothelial Cells by Upregulating TPM1

Abstract

Background: Cerebral cavernous malformations (CCMs) are vascular malformations resulting from the deficiency of cerebral cavernous malformation genes (CCM1, CCM2, and CCM3) in endothelial cells. The genome-wide sequencing of CCM lesions indicated that the level of miRNA-21-5p was aberrantly overexpressed in CCM tissues. Therefore, this study aimed to explore the role of miR-21-5p in CCM. Methods: To establish the in vitro CCM model, CCM1 was silenced in human umbilical vein endothelial cells (HUVECs) by transfecting cells with small interfering RNA (siRNA) for CCM1. The regulatory effects of CCM1-deficiency on miR-21-5p levels were determined using Quantitative Real-Time PCR (qRT-PCR) analysis. The CCM1-siRNA, NC-siRNA, and miR-21-5p-siRNA were respectively delivered into the HUVECs, and the expression levels of the tight junction-related genes (ZO-1 and Claudin 5) were examined using qRT-PCR and Western blot analysis. Furthermore, the cell apoptosis ratio was assessed employing flow cytometry and cell viability was determined using Methyl Thiazolyl Tetrazolium (MTT) assay. Results: It was found that miRNA-21-5p downregulated the expression levels of zonula occludens-1 (ZO-1) and Claudin 5 in CCM (p < 0.05). However, miRNA-21-5p siRNA rescued the reduced expression levels of ZO-1 and Claudin 5 (p < 0.05) and promoted the apoptosis in CCM1-deficient endothelial cells (p < 0.05). Furthermore, the overexpression of tropomyosin 1 (TPM1), as a validated target of miRNA-21-5p, counteracted the miRNA-21-5p-induced cell apoptosis (p < 0.05) and hence upregulated the expression levels of ZO-1 and Claudin 5 (p < 0.05). Conclusions: In summary, the miRNA-21-5p/TPM1 axis was activated in CCM, regulated tight junction-associated proteins, and promoted cell apoptosis in CCM1-deficient endothelial cells. Thus, the miRNA-21-5p/TPM1 axis could be a novel potential target for treating CCM.