LncRNA NEAT1 Promotes Vascular Endothelial Cell Dysfunction via miR-218-5p/GAB2 and Serves as a Diagnostic Biomarker for Deep Vein Thrombosis

Abstract

Objective: Deep vein thrombosis (DVT) is a common peripheral disease. This study aimed to elucidate the diagnostic biomarker of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in the DVT, and explore possible mechanisms in Human umbilical vein endothelial cells (HUVECs).

Methods: 101 patients with lower extremity DVT and 82 healthy controls were enrolled. RT-qPCR was designed to resolve the mRNA levels of NEAT1, miR-218-5p, and GAB2. ROC was applied for the diagnosis of DVT. Systemic inflammation (IL-1β, IL-6, and TNF-α) and adhesion factor (SELP, VCAM-1, and ICAM-1) were examined by the ELISA. And cell proliferation, migration, and apoptosis were conducted by the CCK-8, Transwell, flow cytometry assay. The targeting relationship was validated by Dual luciferase reporter and RIP analysis.

Results: NEAT1 and GAB2 were upregulated in patients with DVT, while miR-218-5p was decreased (P < .01). Serum NEAT1 can identify DVT patients from healthy individuals. NEAT1 was positively correalted with fibrinolysis factors, coagulation factors, and vasoconstrictors. NEAT1 inhibited the proliferation, migration, and promoted apoptosis as well as inflammation and adhesion factors secretion of HUVECs (P < .05), but all were impaired by overexpression of miR-218-5p (P < .05). NEAT1 promoted GAB2 expression in DVT by acting as a sponge for miR-218-5p.

Conclusion: Elevated NEAT1 is a possible DVT diagnostic biomarker, and is implicated in vascular endothelial cell dysfunction via miR-218-5p/GAB2 axis.

Keywords: HUVECs; NEAT1; deep vein thrombosis; diagnostic.

Figure 1.

Serum NEAT1 expression in DVT patients and its diagnostic value. A. RT-qPCR was applicated to explore the expression of NEAT1 in the subjects. B. ROC curves were carried out to evaluate the potential diagnostic capacity of NEAT1. *** P < .001 versus HC group.

Figure 2.

NEAT1 inhibits cell proliferation and migration, promotes apoptosis, and regulates levels of inflammatory and adhesion factors. A. RT-qPCR was applied to resolve NEAT1 levels in HUVECs transfected with si-NEAT1 and pcDNA3.1-NEAT1 plasmids. CCK-8 (B), Transwell (C), and flow cytometry (D) to detect the effects of NEAT1 on cell proliferation, migration, and apoptosis, respectively. ELISA assay was conducted to monitor the effect of NEAT1 on the levels of inflammatory factors (E) and adhesion factors (F). * P < .05, ** P < .01, *** P < .001 versus control.

Figure 3.

NEAT1 as ceRNA competitively binds miR-218-5p in DVT. A. Subcellular localization analysis of NEAT1 in HUVECs. B. ENOCRI predicts and confirmed the potential binding sequence of NEAT1 to miR-218-5p. C. Dual luciferase reporter (DLR) assay indicates that the relative luciferase activity of NEAT1-WT was reduced by a miR-218-3p mimic in HUVECs. D. Association of NEAT1 and miR-218-5p with Ago2 in HUVECs. E. The mRNA levels of miR-218-5p in the serum of patients with DVT were analyzed by RT-qPCR. F. Correlation analysis between serum miR-218-5p and NEAT1 levels. G. RT-qPCR analysis of miR-218-5p expression in HUVECs. The data were presented as the mean ± SD of three independent experiments. *** P < .001 versus control or HC group.

Figure 4.

NEAT1 affects the biological function of HUVECs cells in DVT by regulating mir-218-5p. A. RT-qPCR was applied to resolve miR-218-5p levels in HUVECs transfected with pcDNA3.1-NEAT1 and miR-218-5p mimic plasmids. CCK-8 (B), Transwell (C), and flow cytometry (D) to detect the effects of miR-218-5p and NEAT1 on cell proliferation, migration, and apoptosis, respectively. ELISA assay was conducted to monitor the effect of miR-218-5p on the levels of inflammatory factors (E) and adhesion factors (F). * P < .05, ** P < .01, *** P < .001 versus pcDNA3.1; # P < .05, ## P < .01, ### P < .001 versus mimic NC + pcDNA3.1-NEAT1.

Figure 5.

NEAT1 regulates GAB2 expression through competitive binding of miR-218-5p. A. TargetScan 7.2 predicted the targeting binding sequence between miR-218-5p and GAB2. B. The targeting relationship between miR-218-5p and GAB2 was determined by the Dual luciferase reporting (DLR) assay. C. Enrichment of NEAT1, miR-218-5p, and GAB2 were analyzed by RIP and RT-qPCR assay. D. Serum GAB2 levels in patients with DVT. Correlation of GAB2 levels with miR-218-5p (E) and NEAT1 (F), respectively. G. DLR has conducted the effect of NEAT1 and miR-218-5p on the luciferase activity of GAB2-WT and GAB2-MUT. H. RT-qPCR analysis of the effect of NEAT1 and miR-218-5p levels on GAB2 expression. * P < .05, ** P < .01, *** P < .001 versus control or anti-Ago2 or HC; ## P < .01, ### P < .001 versus mimic NC + pcDNA3.1-NEAT1; &&& P < .05 versus miR-218-5p mimic.