LINC00452 overexpression reverses oxLDL-induced injury of human umbilical vein endothelial cells (HUVECs) via regulating miR-194-5p/IGF1R axis

Abstract

It has been reported that atherosclerosis (AS) is the basis of the development of coronary artery disease (CAD). In addition, a previous study demonstrated that long non-coding RNA LINC00452 was notably downregulated in the whole blood of patients with CAD. However, the role of LINC00452 in the progression of AS remains unclear. Therefore, to mimic AS in vitro, HUVECs were treated with 100 μg/ml oxLDL for 24 h. Reverse transcription-quantitative PCR was performed to detect the expression levels of LINC00452 and IGF1R in HUVECs. Additionally, the cell angiogenetic ability was assessed by tube formation assay, while dual-luciferase reporter assay was carried out to explore the association among LINC00452, miR-194-5p, and IGF1R. The results showed that LINC00452 was downregulated in oxLDL-treated HUVECs. In addition, HUVEC treatment with oxLDL significantly inhibited cell viability, proliferation, and angiogenesis. However, the above effects were all reversed by LINC00452 overexpression. Furthermore, LINC00452 overexpression in HUVECs remarkably inhibited oxLDL-induced cell apoptosis and endothelial to mesenchymal transition. In addition, LINC00452 overexpression could markedly reverse oxLDL-induced inhibition of angiogenesis in HUVEC. The results of dual-luciferase reporter assay indicated that LINC00452 could bind with miR-194-5p. In addition, IGF1R was identified as a downstream target of miR-194-5p. And LINC00452 was able to regulate the miR-194-5p/IGF1R axis in HUVECs. Moreover, LINC00452 overexpression obviously reversed oxLDL-mediated growth inhibition of HUVEC via regulating the miR-194-5p/IGF1R axis. Overall, the current study demonstrated that LINC00452 overexpression reversed oxLDL-induced growth inhibition of HUVECs via regulating the miR-194-5p/IGF1R axis, thus providing a potential beneficial targets for AS.

Keywords: IGF1R; LINC00452; apoptosis; atherosclerosis; miR-194-5p.

Figure 1

LINC00452 overexpression reverses oxLDL-induced growth inhibition of HUVECs. (A) HUVECs were treated with oxLDL (100 μg/mL) for 24 h. RT-qPCR was performed to detect the expression of LINC00452 in HUVECs. (B) HUVECs were transfected with pcDNA3.1-NC or pcDNA3.1-LINC00452 using Lipofectamine 2000 for 24 h. RT-qPCR was performed to detect the efficiency of transfection. (C,D) HUVECs were treated with oxLDL or/and pcDNA3.1-LINC00452. CCK8 and EdU assays were performed to evaluate the viability and proliferation of HUVECs, respectively. **P < 0.01 compared to the control group. ^##P < 0.01 compared to the oxLDL (100 μg/mL) group, n = 3.

Figure 2

LINC00452 overexpression reverses oxLDL-induced apoptosis, migration and EndMT process of HUVECs. HUVECs were treated with oxLDL (100 μg/mL) for 24 h. Next, HUVECs were transfected with pcDNA3.1-LINC00452 using Lipofectamine 2000 for 24 h. (A) Flow cytometry assay was performed to detect the apoptosis of HUVECs. (B) Transwell assay was performed to detect the migration of HUVECs. (C) Western blot assay was performed to detect the expressions of α-SMA, CD31 and VE-cadherin in HUVECs. β-actin was used for normalization. **P < 0.01 compared to control group. ^##P < 0.01 compared to oxLDL (100 μg/mL) group, n = 3.

Figure 3

LINC00452 overexpression reverses oxLDL-induced inhibition of HUVECs angiogenesis. HUVECs were treated with oxLDL (100 μg/mL) for 24 h. Next, HUVECs were transfected with pcDNA3.1-LINC00452 using Lipofectamine 2000 for 24 h. (A) Tube formation assay was performed to measure the branch points and capillary length of HUVECs. (B) Oil-red O staining was used to detect the lipid accumulation in HUVECs. (C) Flow cytometry assay was performed to detect the level of MMP. **P < 0.01 compared to control group. ^##P < 0.01 compared to the oxLDL (100 μg/mL) group, n = 3.

Figure 4

LINC00452 can regulate miR-194-5p/IGF1R axis in HUVECs. HUVECs were treated with oxLDL (100 μg/mL) for 24 h. Next, HUVECs were transfected with pcDNA3.1-LINC00452, miR-194-5p agomir NC, or miR-194-5p agomir using Lipofectamine 2000 for 24 h. (A) Starbase database was used to search the potential targets of LINC00452. (B) Dual-luciferase reporter assay was used to confirm the interaction between LINC00452 and miR-194-5p. (C) The enrichment of miR-194-5p in HUVECs was detected by RNA pull-down. (D) Targetscan database was used to predict the potential targets of miR-194-5p. (E) Dual-luciferase reporter assay was used to confirm the interaction between miR-194-5p and IGF1R. (F) RT-qPCR assay was performed to detect the level of IGF1R. (G–I) Western blot assay was performed to detect the expressions of IGF1R and β-catenin. β-actin was used for normalization. **P < 0.01 compared to control group. ^##P < 0.01 compared to oxLDL (100 μg/mL) group, n = 3.

Figure 5

LINC00452 overexpression reverses oxLDL-induced HUVEC growth inhibition via regulating miR-194-5p/IGF1R pathway. HUVECs were treated with oxLDL (100 μg/mL) for 24 h. Next, HUVECs were transfected with pcDNA3.1-LINC00452 or/and IGF1R siRNA agomir using Lipofectamine 2000 for 24 h. (A) EdU assay was performed to detect the proliferation of HUVECs. (B) Flow cytometry assay was performed to detect the apoptosis of HUVECs. ^##P < 0.01 compared to oxLDL (100 μg/mL) group. ^∧∧P < 0.01 compared to oxLDL+LINC00452 OE group, n = 3.

Figure 6

LINC00452 overexpression reverses oxLDL-induced HUVEC growth inhibition via regulating miR-194-5p/IGF1R pathway. (A) Western blot assay was performed to detect the expressions of IGF1R, β-catenin and VE-cadherin. (B) β-actin was used for normalization. (C) EdU assay was performed to detect the proliferation of HUVECs. **P < 0.01 compared to control group. ^##P < 0.01 compared to oxLDL (100 μg/mL) group. ^∧∧P < 0.01 compared to oxLDL+LINC00452 OE group, n = 3.