This article is mentioned in:

Introduction

Atherosclerosis, a chronic progressive inflammatory disease, is a major cause of morbidity and mortality associated with cardiovascular diseases worldwide (1). The hallmark of atherosclerosis is the accumulation of oxidized low-density lipoprotein (ox-LDL) in the arterial wall, which leads to endothelial dysfunction, chronic inflammation and ultimately plaque formation (2). However, the pathological mechanisms underlying this vascular disease remain to be elucidated.

Endothelial cell senescence is a major driver of atherosclerosis and induces the secretion of pro-inflammatory cytokines and chemokines (3), a phenomenon termed the senescence-associated secretory phenotype (SASP) (4). Among the SASP components, IL-1β, IL-6 and IL-8 are key factors in the local inflammatory milieu of the arterial wall, whereas monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) coordinate the recruitment and adhesion of monocytes and immune cells (5,6). These mediators drive the inflammatory cascade, leukocyte recruitment and adhesion in atherosclerosis, making their targeted inhibition critical for the treatment of this vascular condition.

In recent years, non-coding RNAs such as microRNAs (miRNAs/miRs) and long non-coding RNA (lncRNAs) have been found to play critical roles in regulating the functions of endothelial cells and the pathogenesis of atherosclerosis. In particular, miR-195-5p (miR-195) acts as an inflammatory inducer in various cell types, such as vascular smooth muscle cells (7) and chondrocytes (8), and its overexpression impairs the angiogenesis of human umbilical vein endothelial cells (HUVECs) (9). The inhibition of miR-195 expression has been shown to attenuate atherosclerosis (10). Additionally, the novel lncRNA paternally expressed gene 13 (PEG13) has been shown to function as a competitive endogenous RNA to attenuate neuroinflammation (11) and reduce apoptosis (12). However, the role and mechanism of action of lncRNA PEG13 in atherosclerosis remain to be elucidated.

PI3K/AKT signaling is a key regulator of various cellular processes, including proliferation, inflammation and senescence and has emerged as an important participant in the modulation of endothelial cell function and atherosclerosis progression (13-15). Activation of the PI3K pathway leads to the activation of AKT, which directly phosphorylates the MDM2 proto-oncogene (MDM2) at residues Ser166 and Ser186(16). MDM2 activation promotes endothelial cell survival and attenuates senescence (17), in part by inhibiting tumor protein p53 (p53) and its downstream effector, cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) (18). Additionally, insulin receptor substrate 1 (IRS1) signaling has been identified as an upstream effector of the PI3K/AKT pathway, contributing to the regulation of cellular senescence and inflammation (19,20). Notably, Wang et al (21) showed that IRS1 is a target gene of miR-195.

The present study aimed to elucidate the potential roles of lncRNA PEG13, miR-195, IRS1 and the PI3K/AKT signaling pathway in regulating endothelial cell senescence, SASP and atherosclerosis development. By elucidating the underlying molecular mechanisms, it was hoped to deepen our understanding of the pathophysiology of atherosclerosis and reveal novel therapeutic targets for the development of new strategies for its treatment.

Materials and methods

Screening of mRNA and signaling pathways

The GSE109048 dataset (22) (http://www.ncbi.nlm.nih.gov/geo/) was analyzed using the dataset analysis tool GEO2R (version R 3.2.3; https://www.ncbi.nlm.nih.gov/geo/geo2r/) and the differential expression of mRNAs between tissue samples from healthy donors and those from patients with atherosclerosis was normalized to log2|fold change|>1, with P<0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp/) was used for pathway enrichment analysis of the mRNAs to identify the major pathways involved in atherosclerosis progression.

Cell culture and transfection

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection and cultured in endothelial cell culture medium (cat. no. CM-0122; Procell Life Science & Technology Co., Ltd.). Prior to transfection, the HUVECs were seeded at a density of 2x105 cells/well in a 6-well plate and were exposed to different doses (0, 25, 50, 100 and 200 µg/ml) of ox-LDL (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h. Following transfection, the cells were divided treated with 100 µg/ml of ox-LDL for 24 h to simulate the atherosclerotic environment in vitro. To investigate the role of the PI3K/AKT pathway in ox-LDL-induced HUVEC senescence, these atherosclerotic cells were also incubated with either 10 µM LY294002 (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min to inhibit PI3K signaling or dimethyl sulfoxide (DMSO; Beijing Solarbio Science & Technology Co., Ltd.) as the control group. The lncRNA PEG13 overexpression (ov) vector, overexpression negative control (NC) and miR-195 mimic/inhibitor were purchased from GenePharma (Shanghai, China), and the sequences of the RNAs are listed in Table SI. RNA transfections were performed using Lipofectamine® 3000 according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 4 h. PEG13 overexpression vector and ov-NC were used at a mass of 2 µg/well and miR-195 mimic/inhibitor was used at a dose of 50 nM. At 24 or 48 h post-transfection, cells were collected for gene detection or protein level detection.

Nucleocytoplasmic separation and reverse transcription-quantitative (RT-q)PCR

To investigate the subcellular localization of lncRNA PEG13, a nucleocytoplasmic separation assay was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The cytoplasmic and nuclear fractions were both used for subsequent RNA isolation and RT-qPCR analysis to determine their relative expression levels of lncRNA PEG13. Total RNA was isolated from HUVECs (4x105 cells) using TRIzol® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and RNase-free DNase I (Promega Corporation) was used to digest the genome. The RNA concentration was determined using a BioPhotometer Plus spectrophotometer (Eppendorf Ltd.), following which the RNA was reverse transcribed to complementary DNA (cDNA) using a cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RT-qPCR was performed using the One-Step SYBR Green RT-qPCR Kit (Biomarker Technologies, Inc.) according to the manufacturer's protocol. Primers specific for the target genes (lncRNA PEG13, miR-195 and IRS1) and a reference gene (β-actin or U6) were used (sequences listed in Table SII). The PCR cycling conditions were as follows: an initial denaturation step at 95˚C for 1 min, followed by 40 cycles of denaturation at 95˚C for 12 sec, annealing at 60˚C for 32 sec and extension at 72˚C for 30 sec. The relative expression levels of the target genes were calculated using the 2-ΔΔCq method (23).

RNA pull-down assay

Biotin-labeled lncRNA PEG13, miR-195 and their mutant (mut) probes were synthesized and purified by means of in vitro transcription using the Biotin RNA Labeling Mix (GenePharma) and lysed using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. After pre-clearing with streptavidin magnetic beads (Thermo Fisher Scientific, Inc.), the cell lysate was incubated overnight with the biotin-labeled lncRNA PEG13, miR-195, or mut probes at 4˚C. Streptavidin magnetic beads were then added and the mixture was incubated for 2 h at 4˚C. Subsequently, the beads were thoroughly washed and the bound RNA was eluted and extracted using TRIzol® reagent. The presence of miR-195 or lncRNA PEG13 in the eluted RNA solution was analyzed using RT-qPCR as aforementioned.

Western blotting

After the HUVECs had been lysed with lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), the protein levels were determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. The denatured proteins (20 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidene fluoride membranes (MilliporeSigma). Then, the membranes were rinsed with 10% Tween 20-containing Tris-buffered saline (TBST; Beijing Solarbio Science & Technology Co., Ltd.), blocked with 5% bovine serum albumin (Beijing Solarbio Science & Technology Co., Ltd.) at 22˚C for 1 h, and incubated overnight with primary antibodies against IRS1 (1:1,000; cat. no. MA5-15068; Invitrogen; Thermo Fisher Scientific, Inc.), PI3K (1:1,000; cat. no. ab191606; Abcam), phosphorylated (p)-PI3K (1:500; cat. no. ab182651; Abcam), AKT (1:500; cat. no. ab8805, Abcam), p-AKT (1:500; cat. no. ab38449; Abcam), MDM2 (1:1,000; cat. no. ab259265; Abcam), p53 (1:2,000; cat. no. ab32049; Abcam), p21 (1:3,000; cat. no. ab109520; Abcam) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:3,000; cat. no. ab181602; Abcam) at 4˚C. The membranes were then incubated with horseradish peroxidase-conjugated IgG H&L secondary antibodies (1:2,000; cat. no. ab205718; Abcam) for 2 h at 22˚C. Bands were visualized using Enhanced Chemiluminescence reagent (Thermo Fisher Scientific, Inc.). Densitometry analysis was performed using ImageJ software (version 1.8.0; National Institutes of Health).

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-1β (cat. no. SEKH-0002), IL-6 (cat. no. SEKH-0013) and IL-8 (cat. no. SEKH-0016) in the culture supernatants of HUVECs were measured using ELISA kits (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. In brief, cell debris was first removed from the collected culture supernatants through centrifugation at 1,000 x g for 5 min at 4˚C. ELISA plates were coated with capture antibodies specific for IL-1β, IL-6, or IL-8 and the samples were then added together with the corresponding standards. The absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc.).

Cell proliferation assay

HUVEC proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (4.5x103/well) were seeded in 96-well plates and treated with ox-LDL for 24 h as previously indicated. After treatment, 20 µl of MTT solution (5 mg/ml) was added to each well and the plates were incubated at 37˚C for another 4 h. The medium was then gently aspirated and 150 µl of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader.

Cellular angiogenesis assay

Matrigel (BD Biosciences) was dissolved overnight at 4˚C and then diluted with precooled serum-free medium at a volume ratio of 1:3. Subsequently, 40 µl of the diluted solution was added to each well of a precooled 96-well plate, which was then incubated for 2 h at 37˚C to allow the Matrigel to coagulate. Thereafter, the Matrigel was covered with a 50 µl suspension of HUVECs (2x105 cells/per well) and the number of cell lumens was determined using an inverted microscope (Olympus Corporation).

Cellular senescence assay

Cellular senescence was assessed using a senescence-associated beta-galactosidase (SA-β-gal) staining kit (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. In brief, HUVECs were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min at 22˚C and then incubated overnight with the manufacturer-provided SA-β-gal staining solution in a non-CO2 incubator at 37˚C. The following day, the cells were washed with PBS and observed under a light microscope (Olympus Corporation). Senescent cells were identified by the blue staining of their cytoplasm.

Dual-luciferase reporter assay

The LncBase v3 (https://diana.e-ce.uth.gr/lncbasev3/interactions) and TargetScan 7.2 (https://www.targetscan.org/vert_72/) databases were used to predict the binding sites of miRNAs, lncRNAs and mRNAs. For the dual-luciferase reporter assay, either lncRNA PEG13 (WT and mut) or IRS1 (WT and mut) sequence containing miR-195 binding sites were cloned into a pmirGLO luciferase reporter vector (Promega Corporation). Then, HUVECs were co-transfected with the luciferase reporter vector (WT or mut) and the miR-195 mimic or mimic NC at 37˚C for 4 h using Lipofectamine® 3000. After 48 h, the cells were lysed and their luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation) according to the manufacturer's instructions. The firefly luciferase activity in each group was normalized to the Renilla luciferase activity to account for differences in transfection efficiency.

Statistical analysis

Data are expressed as the mean ± standard deviation and were analyzed using GraphPad Prism 8 (GraphPad Software; Dotmatics). The unpaired Student's t-test was used for comparisons between two groups. One-way or two-way analysis of variance was used to compare multiple groups, followed by Tukey's post-hoc test for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

Ox-LDL dose-dependently inhibited lncRNA PEG13 expression

In the present study, ox-LDL inhibited the viability of HUVECs (Fig. 1A) and the expression of lncRNA PEG13 (Fig. 1B) in a dose-dependent manner. The ox-LDL dose of 100 µg/ml was selected for the subsequent mechanistic analyses. RT-qPCR verified the validity of the PEG13 overexpression vector (Fig. 1C). Subsequently, it was observed that the reduced cell viability (Fig. 1D) and downregulation of PEG13 expression (Fig. 1E) due to ox-LDL induction was reversed by PEG13 overexpression. Moreover, PEG13 overexpression alleviated the decline in angiogenic capacity (Fig. 1F) and the aggravated senescence (Fig. 1G) mediated by ox-LDL. Moreover, the ox-LDL-induced decrease in levels of the SASP-related factors IL-1β, IL-6 and IL-8 was markedly enhanced by PEG13 overexpression (Fig. 1H-J). Additionally, overexpression of this lncRNA enhanced the ox-LDL-induced inhibition of expression of the chemotactic/adhesive molecules MCP-1, VCAM-1 and ICAM-1 (Fig. 1K-M).

Figure 1 Effects of ox-LDL and lncRNA PEG13 on HUVECs. (A) Dose-dependent inhibition of cell viability by ox-LDL, as determined with the MTT assay (*P<0.05 vs. 0 µg/ml). (B) LncRNA PEG13 expression in response to ox-LDL, as determined through RT-qPCR analysis (*P<0.05 vs. 0 µg/ml). (C) Validation of the efficacy of the PEG13 overexpression plasmid. (D) Effect of PEG13 overexpression on cell viability under ox-LDL treatment, as determined with MTT assay. (E) PEG13 expression in PEG13-overexpressing cells, as determined through RT-qPCR analysis. (F) Changes in angiogenic capacity due to PEG13 overexpression, as determined by tube formation assay. Magnification, x200. (G) SA-β-gal staining showing the effect of PEG13 overexpression on ox-LDL-induced senescence. Magnification, x200. (H-J) Levels of SASP-related factors (H) IL-1β, (I) IL-6 and (J) IL-8 following PEG13 overexpression, as measured by ELISA. (K) MCP-1, (L) VCAM-1 and (M) ICAM-1 expression levels following PEG13 overexpression, as determined through RT-qPCR analysis. *P<0.05. ox-LDL, oxidized low-density lipoprotein; lncRNA, long non-coding RNA; HUVECs, human umbilical vein endothelial cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-qPCR, reverse transcription-quantitative PCR; SASP, senescence-associated secretory phenotype; MCP-1, monocyte chemoattractant protein-1; VCAM-1, vascular cell adhesion molecule-1; ICAM-1 and intercellular adhesion molecule-1; ov, overexpression; SA-β-gal, senescence-associated beta-galactosidase; NC, negative control.

miR-195 identified as a potential target of PEG13

The high-level expression of cytoplasmic lncRNA PEG13 was aligned with that of the positive control GAPDH and contrasted with the level of U6 expression (Fig. 2A). Through LncBase database analysis, miR-195 emerged as a potential target of PEG13 owing to its established association with adverse atherosclerosis prognosis (24). The RT-qPCR results showed that ox-LDL increased the miR-195 expression level in a dose-dependent manner (Fig. 2B), but this effect was suppressed by PEG13 overexpression (Fig. 2C). After RT-qPCR confirmation of the efficacy of the synthesized miR-195 mimic/inhibitor (Fig. 2D), dual-luciferase reporter assays were performed. Bioinformatics analysis revealed potential binding sites between lncRNA PEG13 and miR-195 (Fig. 2E) and the dual-luciferase reporter assay confirmed reduced luciferase activity in the cells co-transfected with PEG13-WT and the miR-195 mimic. No significant difference was observed in the interaction between PEG13-mut and the miR-195 mimic or miR-NC (Fig. 2F). In the RNA pull-down assays, miR-195 and PEG13 were enriched in the Bio-PEG13 and Bio-miR-195 groups, respectively, whereas no significant changes were observed in the Bio-mut or Bio-miR-195-mut groups, which further confirmed their binding interaction (Fig. 2G and H). Additionally, the miR-195 mimic/inhibitor had no significant effect on PEG13 expression (Fig. 2I). These results confirmed the direct targeting of miR-195 by lncRNA PEG13.

Figure 2 Interaction between lncRNA PEG13 and miR-195 (A) Subcellular fractionation and RT-qPCR showing lncRNA PEG13 localization. (B) Levels of miR-195 expression in the presence of various ox-LDL doses, as determined through RT-qPCR analysis (*P<0.05 vs. 0 µg/ml). (C) Level of miR-195 expression following PEG13 overexpression, as determined through RT-qPCR analysis. (D) Validation of the synthesized miR-195 mimic/inhibitor. (E) TargetScan prediction of potential binding sites between miR-195 and IRS1. (F) Dual-luciferase reporter assay of the interaction between lncRNA PEG13 and miR-195. (G and H) RNA pull-down assay demonstrating (G) miR-195 enrichment in Bio-PEG13 and (H) PEG13 enrichment in Bio-miR-195. (I) RT-qPCR analysis of the effect of the miR-195 mimic/inhibitor on PEG13 expression. *P<0.05. lncRNA, long non-coding RNA; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; RT-qPCR, reverse transcription-quantitative PCR; lncRNA, long non-coding RNA; WT, wild type; mut, mutant; ov, overexpression; NC, negative control.

IRS1 is involved in the lncRNA PEG13/miR-195 axis

The joint analysis of the TargetScan and GSE109048 datasets revealed the overlap of three genes (Fig. 3A), among which IRS1, a potential miR-195 target, is a known activator of the PI3K/AKT pathway (25). The KEGG analysis indicated that the PI3K/AKT pathway was one of the major signaling pathways involved in atherosclerosis (Fig. 3B), further confirming the rationality of IRS1 involvement. The RT-qPCR assay results showed that ox-LDL decreased IRS1 expression in a dose-dependent manner (Fig. 3C) and this downregulation was ameliorated by PEG13 overexpression (Fig. 3D) and negatively regulated by miR-195 (Fig. 3E). Bioinformatics analysis revealed potential binding sites between IRS1 and miR-195 (Fig. 3F) and the dual-luciferase reporter assay confirmed that the gene is indeed a direct target of this miRNA (Fig. 3G). The three small interfering RNAs (siRNAs) designed to target IRS1 (si-IRS1) inhibited the expression of both the gene (Fig. 3H) and its protein (Fig. 3I) in HUVECs, with siRNA-1 showing the highest inhibition rate in follow-up mechanistic experiments.

Figure 3 Involvement of IRS1 and the PI3K/AKT pathway in the lncRNA PEG13/miR-195 axis. (A) Venn diagram showing potential targets for lncRNA PEG13, determined from the joint analysis of TargetScan and GSE109048 datasets. (B) Bubble map showing genes involved in atherosclerosis-related pathways in the GSE109048 dataset. (C) Levels of IRS1 expression in the presence of various doses of ox-LDL, as determined through RT-qPCR analysis (*P<0.05 vs. 0 µg/ml). (D) Levels of IRS1 expression following PEG13 overexpression, as determined through RT-qPCR analysis. (E) Levels of IRS1 expression after miR-195 mimic/inhibitor treatment, as determined through RT-qPCR analysis. (F) Potential binding sites between IRS1 and miR-195, as determined through bioinformatics analysis. (G) Dual-luciferase reporter assay confirmation of IRS1 as a direct target of miR-195. (H) Levels of IRS1 expression in HUVECs transfected with siRNA, as determined through RT-qPCR analysis. (I) IRS1 protein levels in HUVECs transfected with siRNA, as determined through western blot analysis. *P<0.05. IRS1, insulin receptor substrate 1; lncRNA, long non-coding RNA; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; RT-qPCR, reverse transcription-quantitative PCR; HUVECs, human umbilical vein endothelial cells; siRNA, small interfering RNA; WT, wild type; mut, mutant; ov, overexpression; NC, negative control.

PEG13 overexpression enhanced PI3K/AKT signaling

Although the total protein levels of PI3K and AKT remained unchanged, PEG13 overexpression, acting as an activator of the PI3K/AKT pathway, increased the phosphorylation of PI3K and AKT that had been reduced by ox-LDL (Fig. 4A). Next, the effect of the PEG13/miR-195/IRS1 axis on PI3K/AKT expression was evaluated. The depletion of miR-195 via PEG13 overexpression enhanced the activation of IRS1 protein level and PI3K/AKT signaling, but this effect was partially reversed by the suppression of IRS1 expression (Fig. 4B). As expected, PEG13 overexpression enhanced the positive effects of miR-195 depletion on cell viability (Fig. 4C) and angiogenesis (Fig. 4D), ameliorated senescence (Fig. 4E), reduced the IL-1β, IL-6 and IL-8 levels (Fig. 4F-H) and inhibited the ox-LDL-induced downregulation of MCP-1, VCAM-1 and ICAM-1 expression (Fig. 4I-K) and all these effects were partly reversed by si-IRS1.

Figure 4 Activation of PI3K/AKT signaling by PEG13 overexpression. (A) IRS1, p-PI3K, PI3K, AKT and p-AKT protein levels after PEG13 overexpression, as determined through western blot analysis. (B) IRS1, p-PI3K, PI3K, AKT and p-AKT protein levels under PEG13 overexpression, miR-195 depletion and IRS1 suppression conditions, as determined through western blotting. (C) Combined effects of PEG13 overexpression and miR-195 depletion on cell viability, as determined with the MTT assay. (D) Angiogenesis changes due to PEG13 overexpression and miR-195 depletion, as determined with the tube formation assay. Magnification, x200. (E) SA-β-gal staining showing the effects of PEG13 overexpression and miR-195 depletion on senescence. Magnification, x200. (F) IL-1β, (G) IL-6 and (H) IL-8 protein levels following PEG13 overexpression and miR-195 depletion, as measured with ELISA. Levels of (I) MCP-1, (J) VCAM-1 and (K) ICAM-1 expression following PEG13 overexpression and miR-195 depletion, as determined through RT-qPCR analysis. *P<0.05. IRS1, insulin receptor substrate 1; p-, phosphorylated; miR, microRNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SA-β-gal, senescence-associated beta-galactosidase; MCP-1, monocyte chemoattractant protein-1; VCAM-1, vascular cell adhesion molecule-1; ICAM-1 and intercellular adhesion molecule-1; ov, overexpression; NC, negative control.

PI3K/AKT signaling plays an essential role in cellular senescence

To further investigate the role of the PI3K/AKT pathway in ox-LDL-induced HUVEC senescence, LY294002 was used to inhibit PI3K signaling. As a result, the effects of PEG13 overexpression in promoting MDM2 expression and inhibiting the senescence-associated proteins p53 and p21 were blocked in the presence of LY294002 (Fig. 5). This further indicates that the PI3K/AKT signaling pathway is essential for regulating HUVEC senescence, with PEG13 acting as a key mediator in the process.

Figure 5 Role of the PI3K/AKT pathway in cellular senescence. Western blot analysis of MDM2, p53 and p21 protein levels after LY treatment, showing the impact of the PI3K/AKT pathway inhibition on PEG13 overexpression-induced senescence reduction. *P<0.05. MDM2, MDM2 proto-oncogene; LY, LY294002; p53, tumor protein p53; p21, cyclin-dependent kinase inhibitor 1A (CDKN1A); ov, overexpression; NC, negative control; ox-LDL, oxidized low-density lipoprotein.

Discussion

Atherosclerosis can lead to cardiovascular diseases with high rates of morbidity and mortality, including ischemic stroke, coronary heart disease and myocardial infarction, thus posing a serious threat to human health (26,27). Understanding the role of lncRNAs in regulating gene expression is important for understanding the pathological mechanisms of atherosclerosis (28). In the present study, lncRNA PEG13 was used as an entry point to analyze the effects of its downstream targets and signal transduction pathways on the pathogenesis and progression of atherosclerosis. One of the major findings was that ox-LDL reduced both the viability of HUVECs and their expression of lncRNA PEG13 in a dose-dependent manner and these effects could be partially reversed through PEG13 overexpression, thereby confirming for the first time the involvement of lncRNA PEG13 in the pathogenesis of atherosclerosis. Therefore, the present study also investigated the effect of lncRNA PEG13 on HUVEC senescence and the underlying molecular mechanisms involved.

Consistent with previous research findings, the present results suggested that lncRNA PEG13 serves as a critical modulator of endothelial cell senescence in response to ox-LDL exposure and its overexpression promotes HUVEC viability and ameliorates cellular senescence while downregulating the inflammatory cytokines IL-1β, IL-6 and IL-8 (29,30). Furthermore, the decrease in expression of the chemokines/adhesion molecules MCP-1, VCAM-1 and ICAM-1 implied decreased monocyte recruitment and reduced leukocyte adhesion and infiltration (31,32), which are beneficial for reducing inflammation and limiting atherosclerotic plaque growth and instability, further supporting the anti-senescence and anti-inflammatory effects of lncRNA PEG13 in HUVECs. Moreover, nucleocytoplasmic separation experiments revealed that lncRNA PEG13 is predominantly expressed in the cytoplasm and may regulate cellular senescence through a competitive endogenous RNA mechanism (33).

Using LncBase predictions, dual-luciferase reporter assays and RNA pull-down assays, the present study first demonstrated the targeting of miR-195 by PEG13, a critical regulator of the pathogenesis of atherosclerosis (10). The IRS1/PI3K/AKT signaling pathway is involved in various biological processes, including cell survival, angiogenesis and senescence (34,35). In the present study, IRS1 (an activator of the PI3K/AKT pathway) was found to be a target gene of miR-195, with ox-LDL promoting miR-195 expression and inhibiting IRS1 expression in a dose-dependent manner. The suppression of IRS1 expression counteracted the protective effects of PEG13 and the miR-195 inhibitor and was associated with the deactivation of PI3K/AKT signaling. This is the first confirmation that PEG13 regulates endothelial cell inflammation and senescence through the miR-195/IRS1 axis. Next, LY294002 (a PI3K inhibitor) was used to investigate the role of PI3K/AKT signaling in senescence. Previous studies have shown that this signaling pathway activates MDM2 and leads to the repression of p53 and p21, thereby promoting cell survival and reducing senescence (36,37). The expression of p53 and p21 increases in endothelial cells exposed to various stressors, including ox-LDL, leading to endothelial dysfunction and plaque formation (38,39). In the present study, PEG13, which acted as an activator of PI3K/AKT signaling, upregulated the expression of MDM2 and attenuated the ox-LDL-induced increase in p53 and p21 levels, suggesting that the lncRNA PEG13/miR-195/IRS1/PI3K/AKT axis influences cellular senescence by modulating cell cycle regulators. The inhibition of PI3K signaling by LY294002 also inhibited downstream AKT signaling, resulting in increased expression of the senescence-related factor p53 and its downstream effector p21.

The present study had several limitations. First, within the context of atherosclerosis, HUVECs treated with ox-LDL serve as a mature model. The crux of our pursuit has been to delve deeply into the molecular mechanisms at play within this specific setting. As such, the present study did not branch out to encompass various cell types, such as smooth muscle cells, macrophages, or diverse immune cells. Second, the present study did not investigate co-culturing of HUVECs with other cellular forms. This restricts the holistic understanding of the role that lncRNA PEG13 plays not only in atherosclerosis but also in pertinent in vitro systems. Third was the failure of the present study to conduct transcriptomic analysis, such as RNA-seq, on the ox-LDL treated cells. This would have elucidated any global transcriptional effects stemming from the ox-LDL treatment. Fourth, the present study did not venture into assessing the role of lncRNA PEG13 within animal models, which is instrumental in evaluating its potential as a therapeutic target. Finally, the authors have yet to extensively probe other potential targets and pathways influenced by this lncRNA. These gaps form the cornerstone of objectives for future research endeavors.

In conclusion, the present study demonstrated the protective role of lncRNA PEG13 against ox-LDL-induced HUVEC dysfunction, an effect that involves its regulation of the IRS1/PI3K/AKT signaling pathway via miR-195 depletion and its subsequent influence on MDM2 and senescence-related factors p53 and p21 (Fig. 6). Future research based on these findings may contribute to the development of novel therapeutic strategies targeting lncRNA PEG13 or its associated molecular pathways for the treatment and prevention of atherosclerosis.

Figure 6 lncRNA PEG13 activates the PI3K/AKT signaling pathway via the miR-195/IRS1 axis to promote the expression of MDM2 and inhibit that of age-related protein p53 and its downstream effector p21, leading to a decrease in both cells aging and the senescence-associated secretory phenotype. lncRNA, long non-coding RNA; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; IRS1, insulin receptor substrate 1; MDM2, MDM2 proto-oncogene p53, tumor protein p53; p21, cyclin-dependent kinase inhibitor 1A (CDKN1A).

Supplementary Material

Sequences of IRS1 and miR-195 mimic/inhibitor.

Primer sequences for reverse transcription-quantitative PCR.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Authors' contributions

QH and HZ designed and developed the study methodology. QH, HZ and SY performed the experiments and collected and interpreted the data. QH and HZ drafted the manuscript. QH and HZ confirmed the authenticity of all raw data. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References