LINC00460 Stimulates the Proliferation of Vascular Endothelial Cells by Downregulating miRNA-24-3p

Abstract

Objective: To clarify the effect of LINC00460 on mediating the proliferative ability of vascular endothelial cells (ECs) by targeting microRNA-24-3p (miRNA-24-3p), thus influencing the progression of atherosclerotic diseases.

Methods: Relative levels of LINC00460 and miRNA-24-3p in ECs induced with different doses of ox-LDL (oxidized low density lipoprotein) for different time points were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Influences of LINC00460 and miRNA-24-3p on the viability of ECs were assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay. Through dual-luciferase reporter gene assay, the binding between LINC00460 and miRNA-24-3p was evaluated. At last, rescue experiments were performed to identify the function of the LINC00460/miRNA-24-3p axis in regulating the proliferative ability of ECs.

Results: LINC00460 was upregulated after ox-LDL treatment in a dose- and time-dependent manner. Viability of ECs gradually increased with the prolongation of ox-LDL treatment and the treatment of increased dose. The overexpression of LINC00460 enhanced the viability and EdU-positive rate in ECs treated with ox-LDL. miRNA-24-3p was the direct target of LINC00460, which was negatively regulated by LINC00460. miRNA-24-3p was downregulated with the prolongation of ox-LDL treatment. The overexpression of miRNA-24-3p could reverse the effect of LINC00460 on regulating the proliferative ability of ECs.

Conclusions: LINC00460 regulates the proliferative ability of ECs and thus the occurrence and development of coronary atherosclerotic diseases by targeting miRNA-24-3p.

Figure 1

LINC00460 was upregulated after ox-LDL treatment. (a) CCK-8 assay showed the viability in ECs treated with 0, 1, 10, and 100 μg/mL ox-LDL for 24 h. (b) CCK-8 assay showed the viability in ECs treated with 100 μg/mL ox-LDL for 0, 24, 48, and 72 h. (c) Relative level of LINC00460 in ECs treated with 0, 1, 10, and 100 μg/mL ox-LDL for 24 h. (d) Relative level of LINC00460 in ECs treated with 100 μg/mL ox-LDL for 0, 24, 48, and 72 h.

Figure 2

The overexpression of LINC00460 accelerated the proliferative ability of ECs. (a) Transfection efficacy of pcDNA-LINC00460 in ECs. (b) CCK-8 assay showed the viability in ECs transfected with pcDNA-NC or pcDNA-LINC00460. (c) DAPI-labeled, EdU-labeled, and merged images of ECs transfected with pcDNA-NC or pcDNA-LINC00460.

Figure 3

LINC00460 bound to miR-24-3p. (a) Relative levels of miRNA-24-3p, miRNA-485-5p, miRNA-149-5p, miRNA-662, and miRNA-671-5p in ECs transfected with pcDNA-NC or pcDNA-LINC00460. (b) MiR-24-3p level in ECs treated with 100 μg/mL ox-LDL for 0, 24, 48, and 72 h. (c) Binding sites in promoter regions of LINC00460 and miR-24-3p. (d) Luciferase activity in ECs after cotransfection with miR-24-3p mimic/negative control and LINC00460-WT/LINC00460-MT, respectively. (e) Transfection efficacies of miR-24-3p mimics and miR-24-3p inhibitor. (f) LINC00460 level in ECs transfected with NC, miR-24-3p mimics, or miR-24-3p inhibitor.

Figure 4

The overexpression of miR-24-3p reversed the role of LINC00460 on EC proliferation. ECs were transfected with pcDNA-NC, pcDNA-LINC00460 + miR-24-3p control, or pcDNA-LINC00460 + miR-24-3p mimics. (a) CCK-8 assay showed the viability. (b) DAPI-labeled, EdU-labeled, and merged images of ECs.