Long non-coding RNA NEAT1 inhibits oxidative stress-induced vascular endothelial cell injury by activating the miR-181d-5p/CDKN3 axis

Abstract Endothelial cell (EC) dysfunction induces atherosclerotic coronary heart disease (CHD) development. Recent studies demonstrated that lncRNA NEAT1 mediates multiple biological functions of cells. How NEAT1 regulates EC function is still unclear, so this study explored the role and mechanism of NEAT1 in oxidative stress-induced ECs. The levels of NEAT1 and miR-181d-5p were measured in serum samples from ApoE−/− mice and t-BHP-treated human umbilical vein endothelial cells (HUVECs) by qRT-PCR. The potential role of NEAT1 in viability, migration and apoptosis was analyzed by CCK-8, cell metastasis, flow cytometry, dual-luciferase reporter, RNA immunoprecipitation and Western blot assays using HUVECs overexpressing NEAT1. The expression of NEAT1 was increased, but miR-181d-5p expression was decreased in serum samples from both ApoE−/− mice and t-BHP-treated HUVECs. Overexpression of NEAT1 increased viability, migration and CDKN3 expression but decreased apoptotic rates, caspase-3 activity and miR-181d-5p expression in HUVECs. In addition, NEAT1 acted as a promoter of the proangiogenic capacity of HUVECs by targeting miR-181d-5p/CDKN3. Altogether, these findings indicate that NEAT1 may exert a protective effect on HUVECs by regulating the miR-181d-5p/CDKN3A axis.


Introduction
Cardiovascular disease (CVD) is an aggressive disease with a high morbidity rate worldwide [1][2][3]. Excessive oxidative stress and abnormal inflammatory responses accumulate endothelial cell (EC (ECs) injury [4]. The initial step of CVD is EC dysfunction-induced cardiac microvascular injury [5,6]. Thus, the prevention of oxidative stress-induced EC injury is needed to improve therapeutic strategies needed for improving therapeutic stratagems.
For many years, evidence has shown that lncRNAs are closely related to the pathogenesis of CVD [7,8]. Wang et al. showed that long noncoding RNA AK08838 upregulates the viability of H/R cardiomyocytes via miR-30a [9]. LINC00968 expression is upregulated in coronary artery disease tissues and oxLDL-treated ECs. Overexpression of LINC00968 promotes the proliferation and migration of ECs via targeting miR-9-3p [10]. In uremic patients, increased expression of DKFZP434I0714 is a new potential predictor of worse CV outcomes [11]. Abnormal angiogenesis is connected with the pathogenesis of CVD. The role of lncRNA in angiogenesis and EC dysfunction has also been investigated [12,13]. LncRNA XIST was shown to induce oxLDL-treated EC injury by regulating the miR-320/NOD2 pathway [14]. LncRNA H19 contributes to oxLDL-treated EC injury via miR-let-7/periostin. In conclusion, these results suggest that lncRNA is involved in ox-LDL-induced oxidative stress and inflammation responses in ECs. LncRNA NEAT1 is upregulated and functions as a promoter in human malignancies such as hepatocellular carcinoma [15], cervical cancer [16], ovarian cancer [17] and others [18][19][20]. Several studies have also demonstrated that NEAT1 is involved in EC biological behaviors. NEAT1 expression was shown to be increased in glioma endothelial cells (GECs). Knockdown of NEAT1 enhanced blood-tumor barrier permeability via miR-181d-5p [21]. NEAT1 was shown to play a protective role in oxygen-glucose-deprived brain microvascular endothelial cells [22]. NEAT1 also regulates immune cell functions, and NEAT1 expression was reduced in myocardial infarction patients [23]. However, the biological role of NEAT1 in oxidative stress-treated HUVECs remains largely unknown. Herein, the effects of NEAT1 on the proangiogenic capacity of HUVECs were investigated by gain-of-function assays. Furthermore, the underlying mechanism of NEAT1 in t-BHPtreated HUVECs was explored.

Cell culture
Primary HUVECs were maintained in endothelial growth medium (Lonza, MD, USA) with 10% FBS, 5% L-glutamine, 1% EGF and 1% heparin at 37 C in a 5% CO 2 atmosphere [24]. HUVECs at passages 3-6 were used for the following experiments. Two hundred microliters of t-BHP was used to treat HUVECs for 24 h.

Cell viability analysis
First, HUVECs were transfected with the designated vector and treated with t-BHP (200 lM) for 24 h. HUVEC viability was then assessed using a Cell Counting Kit (CCK)-8 assay according to the manufacturer's instructions. The absorbance (OD) at 450 nm was examined with an ELISA reader.

Cell migration and wound healing assays
First, HUVECs were transfected with the designated vector and treated with t-BHP (200 lM) for 24 h. HUVECs were then added into the upper transwell chambers (Costar, Cambridge, MA). A total of 600 mL of DMEM containing 10% FBS was added to the lower chambers. After incubation for 12 h at 37 C, the cells were stained with 0.1% calcein M. The migrated HUVECs were calculated by a Zeiss confocal microscope.
First, HUVECs were transfected with the designated vector and treated with t-BHP (200 lM) for 24 h. HUVECs were seeded into 6-well culture plates overnight. A scratch was generated with a 200-lL pipette tip. Then, 200 lL of DMEM containing 10% FBS was added to the wells at 37 C for 24 h. The relative gap distance was determined by a light microscope.

Cell apoptosis analysis
First, HUVECs were transfected with the designated vector and treated with t-BHP (200 lM) for 24 h. HUVEC apoptosis was analyzed by flow cytometry (Beckman Coulter, Atlanta, USA) using an Annexin V-FITC/PI apoptosis kit. HUVECs were treated with Annexin V-FITC and PI solution for 15 min following the manufacturer's instructions. HUVEC apoptosis was also measured using a caspase-3 activity kit according to the manufacturer's instructions.
RNA immunoprecipitation and biotin-tagged miR-181d-5p pulldown analysis RNA immunoprecipitation assays were performed using an EZMagna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). HUVECs were lysed with RIP lysis buffer, and cell lysates were then incubated with magnetic beads conjugated with human anti-Ago2 antibody or control mouse IgG (Abcam, Shanghai, China). Total RNA was measured by qPCR analysis.
For the biotin-tagged miR-181d-5p pull-down analysis, HUVECs were transfected with biotin-tagged miR-181d-5p for 48 h. Then, HUVEC lysates were harvested and incubated with Dynabeads TM M-280 Streptavidin at 4 C overnight. RNA binding was measured by qPCR analysis.

Real-time PCR analysis
Total RNA from ApoEÀ/Àmouse serum and t-BHP-treated HUVECs was extracted using TRIzol reagent (Invitrogen). Total RNA was transcribed using PrimeScript TM RT Master Mix (TaKaRa, Dalian, China). Then, real-time PCR experiments were performed using a SYBR V R Premix Ex TaqTM II kit (TaKaRa,Dalian, China) (for NEAT1) and a TaqMan Human MiRNA assay kit (TaKaRa, Dalian, China) (for miR-181d-5p). The relative levels were determined according to the 2 À DDCt method

Western blot analysis
Total protein from HUVECs was harvested by RIPA lysis buffer. Protein samples (30 mg) were separated on 10% SDS-PAGE gels, and the samples were transferred onto PVDF membranes. After blocking with 5% nonfat skim milk, the blots were incubated overnight with primary antibodies at 4 C. The membranes were then treated with horseradish peroxidase-conjugated secondary antibody. The bands were detected using an ECL Plus Detection System (Thermo Scientific, CA, USA).

Animal experiments
ApoEÀ/À mouse (male, 17-22 g) were obtained from the Southern Model Animal Center (Shanghai, China). To determine the effect of NEAT1 on atherosclerosis, ApoEÀ/À mouse were divided randomly into a standard chow diet (control) group and a high-fat diet (HFD) group. The high-fat diet mouse were injected with control lentivirus (pcDNA3.1-NC) or pcDNA3.1-NEAT1 through their tail veins. The control group mouse were fed a standard chow diet, while the HFD group mice were fed a high-fat diet (1.25% cholesterol, 40% fat) for 8 weeks. Food intake and body weight were monitored. Serum samples and tissues were harvested for further investigation.

Determination of serum lipid profile
Serum was harvested by centrifugation at 3000 Â g for 10 min at 4 C and maintained at À80 C. Serum lipid profiles were assessed according to the manufacturer's instructions.

Determination of atherosclerotic plaques
The entire aorta was excised from each mouse and fixed with 4% paraformaldehyde. The lesion areas of the aortas were detected by Oil Red O staining. The atherosclerotic plaque areas are expressed as the percent of the positive plaque area of the whole aorta as measured by Image-Pro Plus.

Statistical analysis
All data are presented as the mean ± standard deviation (SD) and analyzed with GraphPad Prism 5. Comparisons were performed with one-way ANOVA followed by Bonferroni post hoc test. Statistical significance was defined as p < .05.

NEAT1 expression is increased in ApoE2/2 mouse and t-BHP-treated HUVECs
The expression pattern of NEAT1 in HFD-treated ApoEÀ/À mouse aortic plaques and t-BHP-treated HUVECs was measured. The quantitative PCR data showed that NEAT1 expression was increased in HFD-treated ApoEÀ/À mouse aortic plaques and t-BHP-treated HUVECs (Figure 1(A,B)).

Overexpression of NEAT1 increases the proliferation and migration of t-BHP-treated HUVECs
To further elucidate how NEAT1 mediates the proangiogenic capacity of HUVECs, pcDNA3.1-NEAT1 was employed. NEAT1 expression was induced in the pcDNA3.1-NEAT1 transfection group (Figure 2(A)). The CCK-8 assay for viability demonstrated that pcDNA3.1-NEAT1 increased the viability of t-BHPtreated HUVECs (Figure 2(B)). In addition, the contribution of NEAT1 to the migration of t-BHP-treated HUVECs was analyzed by transwell and wound healing assays. As shown in Figure 2(C,D), the migration ability was induced in t-BHPtreated HUVECs by NEAT1 overexpression. These data indicated that NEAT1 could promote the proangiogenic capacity of HUVECs.

Overexpression of NEAT1 inhibits the apoptosis of t-BHP-treated HUVECs
The HUVEC apoptosis rate was analyzed by flow cytometry and caspase-3 activity. The results revealed that the apoptotic rate of t-BHP-treated HUVECs was reduced after treatment with pcDNA3.1-NEAT1 (Figure 2(E)), while t-BHP-treated HUVEC caspase-3 activity was also decreased by NEAT1 overexpression (Figure 2(F)). Altogether, these results indicated that NEAT1 suppresses t-BHP-treated HUVEC apoptosis.

NEAT1 alleviates as mouse atherosclerotic plaque formation
Then, we explored the protective effects of NEAT1 on atherosclerotic lesion formation in AS mouse, and there was no remarkable difference in food intake among the groups after 8 weeks (data not shown). In addition, the body weights of AS mouse were increased, whereas pcDNA3.1-NEAT1 prevented this effect (Figure 6(A)). Moreover, serum levels of TC ( Figure 6(B)), TG (Figure 6(C)), and LDL ( Figure 6(D)) in AS mouse were increased and HDL levels were decreased (Figure 6(E)). Notably, transfection with pcDNA3.1-NEAT1 rescued all of these patterns (Figure 6(B-E)). AS mice had increased atherosclerotic plaque area, whereas pcDNA3.1-NEAT1 alleviated the atherosclerotic lesions in vivo ( Figure  6(F)). After transfection with pcDNA3.1-NEAT1, NEAT1 levels increased, while miR-181d-5p expression was reduced in the thoracic aortic plaques of AS mouse (Figure 6(G,H)). These data suggested that pcDNA3.1-NEAT1 prevented atherosclerotic plaque formation in AS mouse.
Previous reports have shown that NEAT1 participates in several biological events, such as proliferation, polarization invasion and metastasis [31][32][33]. However, the function of NEAT1 in EC dysfunction remains unknown. Our results indicated that AS mouse serum and t-BHP-treated HUVECs showed higher levels of NEAT1. To better understand the effect of NEAT1 on HUVECs, the data revealed that NEAT1 promotes the proangiogenic capacity of HUVECs. LncRNAs perform their functions via several mechanisms, such as transcriptional and post-transcriptional alterations, miRNA sponging, and, mRNA and protein regulation. NEAT1 induces colon cancer progression by binding with miR-495-3p and inducing CDK6 expression [34]. NEAT1 exerts ox-LDL-induced inflammation and oxidative stress via downregulating miR-128 in macrophages [35]. Knockdown of NEAT1 plays a suppressive role in cell immunity during sepsis by inducing the miR-125/MCEMP1 pathway [36]. A bioinformatic analysis showed the potential binding sites between NEAT1 and miR-181d-5p. NEAT1 is located primarily in the cytoplasm. As previously reported, we also found that increased NEAT1 inhibits miR-181d-5p expression in HUVECs. Further data revealed that the miR-181d-5p mimic abrogated the effects of NEAT1 overexpression on t-BHP-treated HUVEC injury. These results demonstrated that NEAT1 might target miR-181d-5p and contribute to the angiogenesis process.
It has been indicated that miR-181d-5p is a suppressor of multiple cell processes [25]. We found that AS mouse serum and t-BHP-treated HUVECs showed lower levels of miR-181d-5p. Furthermore, overexpression of NEAT1 inhibited miR-181d-5p expression. The proangiogenic role of NEAT1 in HUVECs was attenuated by a miR181d-5p mimic and further enhanced by anti-miR-181d-5p. These results suggested that NEAT1 exerts a proangiogenic role in HUVECs via negatively regulating miR-181d-5p.
In conclusion, these findings indicated that NEAT1 expression was increased, whereas miR-181d-5p expression was decreased in AS mouse serum and t-BHP-treated HUVECs. The proangiogenic capacity of NEAT1 in HUVECs was at least partially mediated by miR-181d-5p to regulate CDKN3 expression. These findings highlight the key function of NEAT1 in AS and may provide a potential target for treating AS.