Open Access

Long non‑coding RNA PEG13 regulates endothelial cell senescence through the microRNA‑195/IRS1 axis

  • Authors:
    • Qin Huang
    • Haiwen Zhou
    • Songping Yu
  • View Affiliations

  • Published online on: November 3, 2023
  • Article Number: 584
  • Copyright: © Huang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Atherosclerosis is a chronic inflammatory disease characterized by endothelial dysfunction and plaque formation. The present study aimed to elucidate the pathological role of the long non‑coding RNA (lncRNA) paternally expressed 13 (PEG13) in the onset and progression of atherosclerosis. Specifically, its effects on human umbilical vein endothelial cell (HUVEC) proliferation, angiogenesis, senescence and senescence‑associated secretory phenotype (SASP)‑related factors were investigated using cell proliferation, cellular angiogenesis, β‑galactosidase staining, reverse transcription‑quantitative PCR and enzyme‑linked immunosorbent assays. The results showed that oxidized low‑density lipoprotein (ox‑LDL) inhibited lncRNA PEG13 expression and HUVEC viability in a dose‑dependent manner and PEG13 overexpression partially reversed these effects. Additionally, PEG13 overexpression ameliorated the ox‑LDL‑induced impairment of angiogenesis, cellular senescence and SASP. Furthermore, lncRNA PEG13 directly targeted microRNA (miR/miRNA)‑195‑5p, suppressing the ox‑LDL‑induced upregulation of the miRNA. The gene coding for insulin receptor substrate 1 (IRS1), an activator of the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) signaling pathway, was confirmed as a direct target of miR‑195. PEG13 overexpression attenuated the ox‑LDL‑induced inhibition of IRS1 expression and PI3K/AKT signaling and its protective effects on HUVEC viability, angiogenesis and senescence were partially reversed by small interfering RNAs targeting IRS1. The present study demonstrated that lncRNA PEG13 attenuates ox‑LDL‑induced senescence in HUVECs by modulating the miR‑195/IRS1/PI3K/AKT signaling pathway, suggesting a potential therapeutic target for the treatment of atherosclerosis.


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) ( was analyzed using the dataset analysis tool GEO2R (version R 3.2.3; 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 ( 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 ( and TargetScan 7.2 ( 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.


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.

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.

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.

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.


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.

Supplementary Material

Sequences of IRS1 and miR-195 mimic/inhibitor.
Primer sequences for reverse transcription-quantitative PCR.


Not applicable.


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.



Kong P, Cui ZY, Huang XF, Zhang DD, Guo RJ and Han M: Inflammation and atherosclerosis: Signaling pathways and therapeutic intervention. Signal Transduct Target Ther. 7(131)2022.PubMed/NCBI View Article : Google Scholar


Khatana C, Saini NK, Chakrabarti S, Saini V, Sharma A, Saini RV and Saini AK: Mechanistic insights into the oxidized low-density lipoprotein-induced atherosclerosis. Oxid Med Cell Longev. 2020(5245308)2020.PubMed/NCBI View Article : Google Scholar


Xiang Q, Tian F, Xu J, Du X, Zhang S and Liu L: New insight into dyslipidemia-induced cellular senescence in atherosclerosis. Biol Rev Camb Philos Soc. 97:1844–1867. 2022.PubMed/NCBI View Article : Google Scholar


Vellasamy DM, Lee SJ, Goh KW, Goh BH, Tang YQ, Ming LC and Yap WH: Targeting immune senescence in atherosclerosis. Int J Mol Sci. 23(13059)2022.PubMed/NCBI View Article : Google Scholar


Honda S, Ikeda K, Urata R, Yamazaki E, Emoto N and Matoba S: Cellular senescence promotes endothelial activation through epigenetic alteration, and consequently accelerates atherosclerosis. Sci Rep. 11(14608)2021.PubMed/NCBI View Article : Google Scholar


Wu CM, Zheng L, Wang Q and Hu YW: The emerging role of cell senescence in atherosclerosis. Clin Chem Lab Med. 59:27–38. 2020.PubMed/NCBI View Article : Google Scholar


Xu D, Dai R, Chi H, Ge W and Rong J: Long non-coding RNA MEG8 suppresses hypoxia-induced excessive proliferation, migration and inflammation of vascular smooth muscle cells by regulation of the miR-195-5p/RECK axis. Front Mol Biosci. 8(697273)2021.PubMed/NCBI View Article : Google Scholar


Wang Q, Deng F, Li J, Guo L and Li K: The long non-coding RNA SNHG1 attenuates chondrocyte apoptosis and inflammation via the miR-195/IKK-α axis. Cell Tissue Bank. 24:167–180. 2023.PubMed/NCBI View Article : Google Scholar


Jin J, Wang C, Ouyang Y and Zhang D: Elevated miR-195-5p expression in deep vein thrombosis and mechanism of action in the regulation of vascular endothelial cell physiology. Exp Ther Med. 18:4617–4624. 2019.PubMed/NCBI View Article : Google Scholar


Wang Y, Zhang CX, Ge SL and Gong WH: CTBP1-AS2 inhibits proliferation and induces autophagy in ox-LDL-stimulated vascular smooth muscle cells by regulating miR-195-5p/ATG14. Int J Mol Med. 46:839–848. 2020.PubMed/NCBI View Article : Google Scholar


Gao H, Zhang Y, Xue H, Zhang Q, Zhang Y, Shen Y and Bing X: Long non-coding RNA Peg13 alleviates hypoxic-ischemic brain damage in neonatal mice via miR-20a-5p/XIAP axis. Neurochem Res. 47:656–666. 2022.PubMed/NCBI View Article : Google Scholar


Jiang Y, Wang Y, Sun Y and Jiang H: Long non-coding RNA Peg13 attenuates the sevoflurane toxicity against neural stem cells by sponging microRNA-128-3p to preserve Sox13 expression. PLoS One. 15(e0243644)2020.PubMed/NCBI View Article : Google Scholar


Wang N, Zhang X, Ma Z, Niu J, Ma S, Wenjie W and Chen J: Combination of tanshinone IIA and astragaloside IV attenuate atherosclerotic plaque vulnerability in ApoE(-/-) mice by activating PI3K/AKT signaling and suppressing TRL4/NF-κB signaling. Biomed Pharmacother. 123(109729)2020.PubMed/NCBI View Article : Google Scholar


Duan MX, Zhou H, Wu QQ, Liu C, Xiao Y, Deng W and Tang QZ: Andrographolide protects against HG-induced inflammation, apoptosis, migration, and impairment of angiogenesis via PI3K/AKT-eNOS signalling in HUVECs. Mediators Inflamm. 2019(6168340)2019.PubMed/NCBI View Article : Google Scholar


Cheng HW, Chen YF, Wong JM, Weng CW, Chen HY, Yu SL, Chen HW, Yuan A and Chen JJ: Cancer cells increase endothelial cell tube formation and survival by activating the PI3K/Akt signalling pathway. J Exp Clin Cancer Res. 36(27)2017.PubMed/NCBI View Article : Google Scholar


Chibaya L, Karim B, Zhang H and Jones SN: Mdm2 phosphorylation by Akt regulates the p53 response to oxidative stress to promote cell proliferation and tumorigenesis. Proc Natl Acad Sci USA. 118(e2003193118)2021.PubMed/NCBI View Article : Google Scholar


Wu D and Prives C: Relevance of the p53-MDM2 axis to aging. Cell Death Differ. 25:169–179. 2018.PubMed/NCBI View Article : Google Scholar


Sha JY, Li JH, Zhou YD, Yang JY, Liu W, Jiang S, Wang YP, Zhang R, Di P and Li W: The p53/p21/p16 and PI3K/Akt signaling pathways are involved in the ameliorative effects of maltol on D-galactose-induced liver and kidney aging and injury. Phytother Res. 35:4411–4424. 2021.PubMed/NCBI View Article : Google Scholar


Cao YL, Liu DJ and Zhang HG: MiR-7 regulates the PI3K/AKT/VEGF pathway of retinal capillary endothelial cell and retinal pericytes in diabetic rat model through IRS-1 and inhibits cell proliferation. Eur Rev Med Pharmacol Sci. 22:4427–4430. 2018.PubMed/NCBI View Article : Google Scholar


Li CY, Wang LX, Dong SS, Hong Y, Zhou XH, Zheng WW and Zheng C: Phlorizin exerts direct protective effects on palmitic acid (PA)-induced endothelial dysfunction by activating the PI3K/AKT/eNOS signaling pathway and increasing the levels of nitric oxide (NO). Med Sci Monit Basic Res. 24:1–9. 2018.PubMed/NCBI View Article : Google Scholar


Wang Y, Zhang X, Zou C, Kung HF, Lin MC, Dress A, Wardle F, Jiang BH and Lai L: miR-195 inhibits tumor growth and angiogenesis through modulating IRS1 in breast cancer. Biomed Pharmacother. 80:95–101. 2016.PubMed/NCBI View Article : Google Scholar


Miao M, Cao S, Tian Y, Liu D, Chen L, Chai Q, Wei M, Sun S, Wang L, Xin S, et al: Potential diagnostic biomarkers: 6 Cuproptosis- and ferroptosis-related genes linking immune infiltration in acute myocardial infarction. Genes Immun. 24:159–170. 2023.PubMed/NCBI View Article : Google Scholar


Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar


Teng P, Liu Y, Zhang M and Ji W: Diagnostic and prognostic significance of serum miR-18a-5p in patients with atherosclerosis. Clin Appl Thromb Hemost. 27(10760296211050642)2021.PubMed/NCBI View Article : Google Scholar


Cui F and He X: IGF-1 ameliorates streptozotocin-induced pancreatic β cell dysfunction and apoptosis via activating IRS1/PI3K/Akt/FOXO1 pathway. Inflamm Res. 71:669–680. 2022.PubMed/NCBI View Article : Google Scholar


Falk E: Pathogenesis of atherosclerosis. J Am Coll Cardiol. 47 (8 Suppl):C7–C12. 2006.PubMed/NCBI View Article : Google Scholar


Herrington W, Lacey B, Sherliker P, Armitage J and Lewington S: Epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease. Circ Res. 118:535–546. 2016.PubMed/NCBI View Article : Google Scholar


Simion V, Zhou H, Haemmig S, Pierce JB, Mendes S, Tesmenitsky Y, Pérez-Cremades D, Lee JF, Chen AF, Ronda N, et al: A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus. Nat Commun. 11(6135)2020.PubMed/NCBI View Article : Google Scholar


Jiang YH, Jiang LY, Wang YC, Ma DF and Li X: Quercetin attenuates atherosclerosis via modulating oxidized LDL-induced endothelial cellular senescence. Front Pharmacol. 11(512)2020.PubMed/NCBI View Article : Google Scholar


Cao H, Jia Q, Yan L, Chen C, Xing S and Shen D: Quercetin suppresses the progression of atherosclerosis by regulating MST1-mediated autophagy in ox-LDL-induced RAW264.7 macrophage foam cells. Int J Mol Sci. 20(6093)2019.PubMed/NCBI View Article : Google Scholar


Zhang Q, Liu J, Duan H, Li R, Peng W and Wu C: Activation of Nrf2/HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress. J Adv Res. 34:43–63. 2021.PubMed/NCBI View Article : Google Scholar


Malekmohammad K, Sewell RDE and Rafieian-Kopaei M: Antioxidants and atherosclerosis: Mechanistic aspects. Biomolecules. 9(301)2019.PubMed/NCBI View Article : Google Scholar


Liu Y, Liu N and Liu Q: Constructing a ceRNA-immunoregulatory network associated with the development and prognosis of human atherosclerosis through weighted gene co-expression network analysis. Aging (Albany NY). 13:3080–3100. 2021.PubMed/NCBI View Article : Google Scholar


Kim W, Noh H, Lee Y, Jeon J, Shanmugavadivu A, McPhie DL, Kim KS, Cohen BM, Seo H and Sonntag KC: MiR-126 regulates growth factor activities and vulnerability to toxic insult in neurons. Mol Neurobiol. 53:95–108. 2016.PubMed/NCBI View Article : Google Scholar


Perluigi M, Pupo G, Tramutola A, Cini C, Coccia R, Barone E, Head E, Butterfield DA and Di Domenico F: Neuropathological role of PI3K/Akt/mTOR axis in down syndrome brain. Biochim Biophys Acta. 1842:1144–1153. 2014.PubMed/NCBI View Article : Google Scholar


Huang L, Ye Q, Lan C, Wang X and Zhu Y: AZD6738 Inhibits fibrotic response of conjunctival fibroblasts by regulating checkpoint kinase 1/P53 and PI3K/AKT pathways. Front Pharmacol. 13(990401)2022.PubMed/NCBI View Article : Google Scholar


Safi A, Heidarian E and Ahmadi R: Quercetin synergistically enhances the anticancer efficacy of docetaxel through induction of apoptosis and modulation of PI3K/AKT, MAPK/ERK, and JAK/STAT3 signaling pathways in MDA-MB-231 breast cancer cell line. Int J Mol Cell Med. 10:11–22. 2021.PubMed/NCBI View Article : Google Scholar


Lyu TJ, Zhang ZX, Chen J and Liu ZJ: Ginsenoside Rg1 ameliorates apoptosis, senescence and oxidative stress in ox-LDL-induced vascular endothelial cells via the AMPK/SIRT3/p53 signaling pathway. Exp Ther Med. 24(545)2022.PubMed/NCBI View Article : Google Scholar


Liu Y, Jia L, Min D, Xu Y, Zhu J and Sun Z: Baicalin inhibits proliferation and promotes apoptosis of vascular smooth muscle cells by regulating the MEG3/p53 pathway following treatment with ox-LDL. Int J Mol Med. 43:901–913. 2019.PubMed/NCBI View Article : Google Scholar

Related Articles

Journal Cover

Volume 26 Issue 6

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
Spandidos Publications style
Huang Q, Zhou H and Yu S: Long non‑coding RNA <em>PEG13</em> regulates endothelial cell senescence through the microRNA‑195/IRS1 axis. Exp Ther Med 26: 584, 2023
Huang, Q., Zhou, H., & Yu, S. (2023). Long non‑coding RNA <em>PEG13</em> regulates endothelial cell senescence through the microRNA‑195/IRS1 axis. Experimental and Therapeutic Medicine, 26, 584.
Huang, Q., Zhou, H., Yu, S."Long non‑coding RNA <em>PEG13</em> regulates endothelial cell senescence through the microRNA‑195/IRS1 axis". Experimental and Therapeutic Medicine 26.6 (2023): 584.
Huang, Q., Zhou, H., Yu, S."Long non‑coding RNA <em>PEG13</em> regulates endothelial cell senescence through the microRNA‑195/IRS1 axis". Experimental and Therapeutic Medicine 26, no. 6 (2023): 584.