- Email: [email protected]
IGF2 axis in ox-LDL-stimulated VSMC and HUVEC
Life Sciences 243 (2020) 117287
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
LncRNA TUG1 regulates proliferation and apoptosis by regulating miR148b/IGF2 axis in ox-LDL-stimulated VSMC and HUVEC
T
⁎
Xiaoguang Wu, Xiaohui Zheng , Jing Cheng, Kai Zhang, Cao Ma Department of Emergency, Fuwai Central China Cardiovascular Hospital, Central China Fuwai Hospital of Zhengzhou University, Henan Provincial People's Hospital, Zhengzhou, Henan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherosclerosis TUG1 miR-148b IGF2 VSMC HUVEC
Vascular smooth muscle cell (VSMC) accumulation and endothelial cell dysfunction are associated with pathogenesis of atherosclerosis. Long noncoding RNA taurine up-regulated gene 1 (TUG1) has been reported to play an important role in cardiovascular diseases, including atherosclerosis. However, the regulatory mechanism underlying TUG1 in atherosclerosis is far from understood. VSMC and human umbilical vein endothelial cells (HUVEC) stimulated by oxidized low-density lipoprotein (ox-LDL) were used as cellular model of atherosclerosis. Cell proliferation and apoptosis were detected by CCK-8, flow cytometry and Western blot. The expression levels of TUG1, microRNA (miR)-148b and insulin-like growth factor 2 (IGF2) were measured by quantitative real-time polymerase chain reaction or Western blot. The target association among TUG1, miR-148b and IGF2 was determined by luciferase reporter assay and RNA immunoprecipitation. The expression of TUG1 was increased in ox-LDL-treated VSMC and HUVEC. Silence of TUG1 inhibited proliferation and promoted apoptosis in ox-LDLtreated VSMC but induced proliferation promotion and apoptosis inhibition in HUVEC stimulated by ox-LDL. miR-148b was a target of TUG1 and its knockdown reversed the effect of TUG1 silence on proliferation and apoptosis of VSMC and HUVEC challenged by ox-LDL. IGF2 was a target of miR-148b and miR-148b regulated proliferation and apoptosis in ox-LDL-treated VSMC and HUVEC by targeting IGF2. TUG1 promoted IGF2 protein expression by sponging miR-148b. TUG1 knockdown attenuated ox-LDL-induced injury through regulating proliferation and apoptosis of VSMC and HUVEC by miR-148b/IGF2 axis, providing a novel mechanism for pathogenesis of atherosclerosis.
1. Introduction Atherosclerosis is a common cardiovascular disease with high morbidity and mortality [1]. The aberrant proliferation of vascular smooth muscle cells (VSMC) is associated with progression of early atherosclerosis and vascular injury [2]. Moreover, endothelial cell injury is a key contributor for cardiovascular diseases, including atherosclerosis [3]. Oxidized low-density lipoprotein (ox-LDL) accumulation is one of the major risks of atherosclerosis, which contributes to the accelerated atherosclerosis [4,5]. However, the mechanism underlying how ox-LDL regulates proliferation and apoptosis of VSMC and human umbilical vein endothelial cells (HUVEC) remains largely unclear. Long noncoding RNAs (lncRNAs) with over 200 nucleotides are involved in regulation of cardiovascular diseases by competing endogenous RNA (ceRNA) networks of lncRNA/microRNA (miRNA)/ mRNA axis [6]. Moreover, lncRNAs exhibit essential roles in
atherosclerosis by regulating lipid metabolism, inflammatory response, cell proliferation, apoptosis and migration [7]. Meanwhile, lncRNAs are dysregulated by ox-LDL treatment and associated with the function of VSMC and endothelial cells [8–10]. Taurine up-regulated gene 1 (TUG1) is a functional lncRNA having a vital role in human cancers and diseases [11–13]. In addition, TUG1 could promote macrophages growth and inflammatory response in atherosclerosis [14]. Besides, previous studies indicate that TUG1 could promote atherosclerosis by increasing VSMC proliferation and triggering endothelial cell apoptosis [14–16]. However, the mechanism underlying TUG1 in atherosclerosis has not been fully understood. miRNAs which could be sponged by lncRNAs have important roles in regulation of atherosclerosis development [17]. miR-148b has been reported as an inhibitor of atherosclerosis by decreasing VSMC proliferation and migration but increasing apoptosis [18,19]. Furthermore, insulin-like growth factor (IGF) system is responsible for the function of
⁎ Corresponding author at: Department of Emergency, Central China Fuwai Hospital of Zhengzhou University, Fuwai Central China Cardiovascular Hospital, Henan Provincial People's Hospital, No.1 Fuwai Road, Zhengzhou 451464, Henan, China. E-mail address: [email protected] (X. Zheng).
https://doi.org/10.1016/j.lfs.2020.117287 Received 12 October 2019; Received in revised form 30 December 2019; Accepted 7 January 2020 Available online 08 January 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 1. The expression of TUG1 is increased in VSMC and HUVEC stimulated by ox-LDL. (A and B) qRT-PCR was performed to detect the expression of TUG1 in VSMC and HUVEC treated by ox-LDL. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group (non-treated with ox-LDL group).
Fig. 2. Knockdown of TUG1 regulates proliferation and apoptosis in VSMC and HUVEC stimulated by ox-LDL. (A) The abundance of TUG1 was measured in VSMC and HUVEC after transfection of si-TUG1 or si-NC in the presence of ox-LDL or not. Cell viability (B and C), apoptosis (D) and protein levels of Bax, Bcl-2 and PCNA (E) were determined in VSMC and HUVEC transfected with si-TUG1 or si-NC after treatment of ox-LDL or not. **P < 0.01, ***P < 0.001, compared with si-NC group or ox-LDL + si-NC group.
two cell lines. Additionally, we explored the potential regulatory network of TUG1/miR-148b/IGF2 axis.
endothelial cells in vascular diseases [20]. IGF2 is one member of IGF family, which is involved in regulation of cell proliferation, migration and survival and associated with cardiovascular diseases [21]. Intriguingly, starBase database predicted that TUG1 and IGF2 shared the complementary binding sites of miR-148b, which stimulated us to assume the ceRNA network of TUG1/miR-148b/IGF2. In this study, VSMC and HUVEC were stimulated by ox-LDL as previous studies [22–25]. Moreover, we measured the expression of TUG1 and investigated the effect of TUG1 on cell proliferation and apoptosis in the
2. Materials and methods 2.1. Cell culture, ox-LDL treatment and transfection The human VSMC from human umbilical artery and HUVEC were purchased from BeNa Culture Collection (Beijing, China). VSMC and 2
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 3. TUG1 is a decoy of miR-148b. (A) starBase online predicted the complementary sequences between TUG1 and miR-148b. (B and C) Luciferase activity was analyzed in VSMC and HUVEC co-transfected with miR-NC or miR-148b and TUG1-WT or TUG1-MUT. (D and E) RIP assay was performed in VSMC and HUVEC transfected with miR-148 or miR-NC and TUG1 level was detected by qRT-PCR. (F) qRT-PCR was used to measure the level of miR-148b in VSMC and HUVEC after treatment of ox-LDL. (G and H) The expression of miR-148b was examined in VSMC and HUVEC transfected with pcDNA, TUG1, si-NC or si-TUG1. **P < 0.01, ***P < 0.001, compared with miR-NC group for B-E, with control group for F, with pcDNA or si-NC group for G and H.
2.2. CCK-8
HUVEC were cultured in DMEM (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum and antibiotics and maintained at 37 °C with 5% CO2. For construct of atherosclerosis model in vitro, VSMC and HUVEC were stimulated with 100 μg/ml ox-LDL (Solarbio, Beijing, China) [22–25]. The overexpression vectors of TUG1 and IGF2 were generated by inserting into pcDNA3.1 empty vector (pcDNA) (Thermo Fisher Scientific, Wilmington, DE, USA). siRNA against TUG1 (si-TUG1) ( 5′-GGATATAGCCAGAGAACAA-3′), siRNA negative control (si-NC) ( 5′-GTTCTCCGAACGTGTCACGT-3′), miR-148b mimic (miR-148b) ( 5′-UCAGUGCAUCACAGAACUUUGU-3′), miRNA negative control (miRNC) (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-148b inhibitor (antimiR-148b) (5′-ACAAAGUUCUGUGAUGCACUGA-3′) and inhibitor negative control (anti-miR-NC) (5′-CAGUACUUUUGUGUAGUACAA-3′) were generated by GenePharm (Shanghai, China). VSMC and HUVEC were transfected with constructed oligonucleotides at 30 nM concentration using Lipofectamine 2000 (Thermo Fisher Scientific) for 24 h. Subsequently, transfected cells were collected for further experiments.
Transfected or non-transfected VSMC and HUVEC (3000 cells/well) were seeded into 96-well plates in triplicate and then incubated with ox-LDL. After 0, 24, 48 and 72 h, 10 μl CCK-8 solution (Beyotime, Shanghai, China) was added into each well and cells were continuously cultured for 3 h. The optical density value of each well at 450 nm was measured by microplate reader (Bio-Rad, Hercules, CA, USA). 2.3. Flow cytometry VSMC and HUVEC were stimulated with ox-LDL for 72 h. The cells were collected and incubated in binding buffer, followed by staining with Annexin V-FITC and PI solution in Annexin V-FITC Apoptosis Detection kit (Beyotime). The apoptotic cells were measured by a flow cytometer and the apoptotic rate of VSMC and HUVEC at early and late apoptosis was presented as the percentage of cells with Annexin V-FITC staining positive and PI staining negative or positive. 2.4. Western blot ProteoPrep® Total Extraction Sample Kit and Bicinchoninic Acid Kit 3
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 4. Inhibition of miR-148b reverses the effect of TUG1 knockdown on proliferation and apoptosis in VSMC and HUVEC stimulated by ox-LDL. (A and B) The expression of miR-148b was detected in VSMC and HUVEC after transfection of si-NC, si-TUG1, si-TUG1 + anti-miR-NC or anti-miR-148b. Cell viability (C and D), apoptosis (E and F) and protein abundances of Bax, Bcl-2 and PCNA (G and H) were determined in VSMC and HUVEC transfected with si-NC, si-TUG1, siTUG1 + anti-miR-NC or anti-miR-148b after treatment of ox-LDL. **P < 0.01, ***P < 0.001, compared with si-NC or si-TUG1 + anti-miR-NC group.
TUG1, IGF2 and miR-148b were analyzed by 2−ΔΔCt method [26].
(Sigma) were used for extraction and concentration determination of total protein from VSMC and HUVEC. The protein lysates (30 μg) were separated by SDS-PAGE and then electro-transferred to PVDF membranes (Millipore, Billerica, MA, USA). Following the block using 5% non-fat milk, the membranes were incubated with primary antibodies and secondary antibody, including anti-Bax (ab77566, 1:1000 dilution, 21 kDa), anti-Bcl-2 (ab201566, 1:2000 dilution, 26 kDa), anti-PCNA (ab220208, 1:2000 dilution, 29 kDa), anti-IGF2 (ab63294, 1:1000 dilution, 50 kDa) and HRP-conjugated IgG (ab6728, 1:8000 dilution), which were purchased from Abcam (Cambridge, MA, USA). Moreover, anti-β-actin (ab49900, 1:30,000 dilution, 42 kDa, Abcam) was used as loading control. The protein blot signaling was visualized using enhanced chemiluminescence solution (Beyotime) and film in the dark.
2.6. Luciferase reporter assay and RNA immunoprecipitation (RIP) The potential complementary sequences of miR-148b and TUG1 or IGF2 were predicted by starBase online (http://starbase.sysu.edu.cn/). The wild-type luciferase reporter vectors of TUG1 and IGF2 (TUG1-WT and IGF2-WT) were generated by inserting the sequences of TUG1 or IGF2 containing miR-148b binding sites into pmirGLO luciferase reporter vector (Promega, Madison, WI, USA). Their corresponding mutants (TUG1-MUT and IGF2-MUT) were obtained by mutating the seed sites of miR-148b. VSMC and HUVEC were transfected with TUG1-WT, TUG1-MUT, IGF2-WT or IGF2-MUT, along with miR-148b or miR-NC. The luciferase reporter assay was carried out using luciferase activity analysis kit (Promega) after the transfection for 24 h. RIP assay was performed in VSMC and HUVEC transfected with miR-148b or miR-NC using Magna RNA immunoprecipitation kit (Millipore) in accordance with the manufacturer's protocols. In brief, the lysed cells were incubated with RIP buffer with magnetic beads conjugated by anti-Ago2 (ab32381, Abcam) or IgG (AP112, Sigma). The enriched abundances of TUG1 and IGF2 by RIP were determined by qRT-PCR.
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) A total of 1 μg RNA isolated using Trizol reagent (Thermo Fisher Scientific) was reversely transcribed to cDNA with the special RT-PCR Kit (Thermo Fisher Scientific) following the manufacturer's instructions. The synthesized cDNA was diluted by 1:5 and then qRT-PCR was performed on ABI 7500 Real-time PCR system (Bio-Rad) with SYBR Green mix (Thermo Fisher Scientific) and primers: TUG1 (Forward, 5′-TAGC AGTTCCCCAATCCTTG-3′; Reverse, 5′-CACAAATTCCCATCATTC CC3′); IGF2 (Forward, 5′-AGACCCTTTGCGGTGGAGA-3′; Reverse, 5′-GG AAACATCTCGCTCGGACT-3′); miR-148b(Forward, 5′-ACACTCCAGCT GGGT CAGTGCATC-3′; Reverse, 5′-CTCAACTGGTGTCGTGGA-3′). GAPDH (Forward, 5′-CAGCCTCAAGATCATCAGCA-3′; Reverse, 5′-TGT GGTCATGAGTCCTT CCA-3′) and U6 (Forward, 5′-CGCTTCGGCAGCA CATATAC-3′; Reverse, 5′-TTCACGAATTTGCGTGTCAT-3′) were used as internal control for RNA expression. The relative expression levels of
2.7. Statistical analysis The experiments were repeated three times with three replicates. The data were presented as mean ± standard deviation and analyzed by GraphPad Prism 7.0. The difference between two or more groups was compared by Student's t-test or ANOVA with Tukey's post hoc test. It was regarded as statistically significant when P < 0.05 (*P < 0.05, 4
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 5. IGF2 is a target of miR-148b. (A) The binding sites of miR-148b and IGF2 were provided by starBase online. (B-E) Luciferase reporter and RIP assays were performed in VSMC and HUVEC transfected with miR-148b or miR-NC to validate the association between miR-148b and IGF2. (F) The protein level of IGF2 was measured in VSMC and HUVEC after stimulation of ox-LDL by Western blot. (G and H) Western blot assay was carried out to detect the level of IGF2 protein in VSMC and HUVEC transfected with miR-NC, miR-148b, anti-miR-NC or anti-miR-148b. **P < 0.01, ***P < 0.001, compared with miR-NC group for B-E, with control group for F, with miR-NC or anti-miR-NC group for G and H.
decreased viability of VSMC and increased viability of HUVEC in the presence of ox-LDL at 48 and 72 h (Fig. 2B and C). Additionally, the analysis of flow cytometry described that down-regulation of TUG1 notably enhanced apoptosis in VSMC but reduced the apoptosis in HUVEC stimulated by ox-LDL (Fig. 2D). What's more, Bax protein level was increased but Bcl-2 and PCNA protein abundances were decreased in VSMC treated by ox-LDL, while these events were opposite in oxLDL-treated HUVEC (Fig. 2E).
**P < 0.01, ***P < 0.001). 3. Results 3.1. TUG1 expression is enhanced in ox-LDL-treated VSMC and HUVEC The cellular model of atherosclerosis was constructed using VSMC and HUVEC stimulated by ox-LDL. As shown in Supplementary Fig. 1A–D, treatment of ox-LDL promoted VSMC viability and inhibited apoptosis, while it decreased viability and increased apoptosis in HUVECs. Moreover, the expression level of TUG1 was measured in VSMC and HUVEC treated via different concentrations of ox-LDL. Results showed that TUG1 expression was evidently elevated in VSMC and HUVEC after stimulation of ox-LDL in a concentration dependent manner (Fig. 1A and B). 100 μg/ml ox-LDL that induced extremely significant up-regulation of TUG1 (P < 0.001) was used for further experiments.
3.3. TUG1 is a decoy of miR-148b To explore the underlying mechanism addressed by TUG1, starBase database was used to determine the putative complementary sequences between TUG1 and miR-148b at position: chr22: 31372748–31372770 (Fig. 3A). To validate their association, the constructed luciferase reporter vectors TUG1-WT or TUG1-MUT were transfected into VSMC and HUVEC. The luciferase reporter assay displayed that miR-148b addition led to great loss of luciferase activity in VSMC and HUVEC transfected with TUG1-WT, whereas it showed little effect in TUG1MUT group (Fig. 3B and C). Moreover, overexpression of miR-148b induced higher enrichment level of TUG1 by Ago2 RIP in VSMC and HUVEC (Fig. 3D and E). In ox-LDL-challenged cells, the expression of miR-148b was specially decreased in comparison to control group (Fig. 3F). Additionally, the abundance of miR-148b in VSMC and
3.2. Silence of TUG1 regulates proliferation and apoptosis in ox-LDLtreated VSMC and HUVEC To investigate the role of TUG1 in atherosclerosis, its abundance was knocked down in VSMC and HUVEC by using si-TUG1, which was confirmed in Fig. 2A. Furthermore, interference of TUG1 obviously 5
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 6. IGF2 attenuates the effect of miR-148b on proliferation and apoptosis in VSMC and HUVEC stimulated by ox-LDL. (A and B) Western blot was conducted to determine the protein level of IGF2 in VSMC and HUVEC after transfection of miR-NC, miR-148b, pcDNA, IGF2, miR-148b + pcDNA or IGF2. Cell viability (C and D), apoptosis (E and F) and protein levels of Bax, Bcl-2 and PCNA (G and H) were measured in VSMC and HUVEC transfected with miR-NC, miR-148b, miR-148b and pcDNA or IGF2 after treatment of ox-LDL. **P < 0.01, ***P < 0.001, compared with miR-NC, pcDNA or miR-148b + pcDNA group.
VSMC and HUVEC after treatment of ox-LDL compared with that in control group (Fig. 5F). Besides, IGF2 protein level was significantly reduced via miR-148b addition and increased by miR-148b deletion in the cells (Fig. 5G and H).
HUVEC was markedly decreased by overexpressing TUG1 and increased by silencing TUG1 (Fig. 3G and H). 3.4. TUG1 regulates proliferation and apoptosis by sponging miR-148b in ox-LDL-treated VSMC and HUVEC
3.6. miR-148b regulates proliferation and apoptosis by targeting IGF2 in oxLDL-treated VSMC and HUVEC
To explore whether TUG1-mediated regulation of atherosclerosis progression was regulated by miR-148b, VSMC and HUVEC were transfected with si-NC, si-TUG1, si-TUG1 and anti-miR-NC or anti-miR148b. As displayed in Fig. 4A and B, the abundance of miR-148b promoted by TUG1 silence was decreased by transfection of anti-miR-148b in VSMC and HUVEC. Moreover, deficiency of miR-148b reversed silencing TUG1-induced viability inhibition in VSMC and viability promotion in HUVEC treated with ox-LDL (Fig. 4C and D). Meanwhile, inhibition of miR-148b weakened the cell apoptosis induced by TUG1 knockdown in ox-LDL-treated VSMC and attenuated the apoptosis suppression caused via TUG1 silence in ox-LDL-stimulated HUVEC (Fig. 4E and F). Furthermore, the effect of TUG1 interference on protein levels of Bax, Bcl-2 and PCNA in VSMC and HUVEC challenged by oxLDL was abolished via miR-148b exhaustion (Fig. 4G and H).
To explore whether IGF2 was involved in miR-148b-mediated regulatory mechanism, VSMC and HUVEC were transfected with miR-NC, miR-148b, pcDNA, IGF2, miR-148b + pcDNA or IGF2. After the transfection, the protein level of IGF2 was inhibited by miR-148b addition, and restored by introduction of IGF2 in VSMC and HUVEC (Fig. 6A and B). Furthermore, overexpression of miR-148b significantly repressed the viability of VSMC treated by ox-LDL and increased the viability of HUVEC stimulated by ox-LDL at 72 h (Fig. 6C and D). Additionally, addition of miR-148b remarkably promoted or suppressed apoptosis in VSMC or HUVEC treated by ox-LDL, respectively (Fig. 6E and F). miR-148b overexpression increased Bax protein and reduced Bcl-2 and PCNA protein levels in VSMC treated by ox-LDL and reduced Bax level and increased Bcl-2 and PCNA protein levels in ox-LDLchallenged HUVEC (Fig. 6G and H). However, IGF2 overexpression led to opposite effect on these events and weakened the role of miR-148 (Fig. 6C–H).
3.5. IGF2 is a target of miR-148b In order to further elucidate the regulatory mechanism, the target of miR-148b was searched by starBase database, which predicted the binding sites of miR-148b and IGF2 at position: chr11:2151600–2151605 (Fig. 5A). Moreover, luciferase reporter assay was used to validate this prediction and results showed that in IGF2-WT group, transfection of miR-148b resulted in obvious reduction of luciferase activity in VSMC and HUVEC, while it did not affect the activity when mutating the seed sites in IGF2-MUT group (Fig. 5B and C). Meanwhile, the level of IGF2 enriched by Ago2 RIP was higher in the cells transfected with miR-148b than that in miR-NC group (Fig. 5D and E). Furthermore, the protein level of IGF2 was markedly enhanced in
3.7. TUG1 regulates IGF2 expression by sponging miR-148b To validate the interaction between TUG1 and IGF2, the luciferase reporter assay was performed. As shown in Fig. 7A and B, the luciferase activity was greatly decreased by overexpression of miR-148a in VSMC and HUVEC transfected with IGF2-WT luciferase reporter vector. In addition, introduction of TUG1 evidently increased the luciferase activity and abolished the suppressive role of miR-148b. which was abated by introduction of TUG1. Moreover, the protein level of IGF2 in 6
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 7. TUG1 regulates IGF2 expression by targeting miR-148b. (A and B) Luciferase activity was determined in VSMC and HUVEC transfected with IGF2-WT, IGF2-WT + miR-148b, IGF2-WT + TUG1, IGF2-WT + miR-148b + pcDNA or IGF2WT + miR-148b + TUG1. (C) Western blot was conducted to detect the protein level of IGF2 in VSMC transfected with pcDNA, TUG1, TUG1 and miR-NC or miR-148b. (D) The protein level of IGF2 was measured in HUVEC transfected with si-NC, siTUG1, si-TUG1 and anti-miR-NC or anti-miR-148b. **P < 0.01, ***P < 0.001, compared with IGF2-WT or IGF2-WT + miR-148b + pcDNA group for A and B, with pcDNA or TUG1 + miR-NC group for C, with si-NC or si-TUG1 + anti-miR-NC group for D.
damage tolerance and attenuates apoptosis [34,35]. By measurement of cell viability, apoptosis and protein levels of Bax, Bcl-2 and PCNA, VSMC proliferation and HUVEC apoptosis were induced by ox-LDL treatment, indicating that the cellular model of atherosclerosis was corroborated. Moreover, we found that ox-LDL exposure increased TUG1 abundance in the cells, which reflected that high expression of TUG1 might be a contributor of atherosclerosis development. Furthermore, loss-of-function experiments revealed that TUG1 knockdown attenuated VSMC proliferation and HUVEC apoptosis induced via ox-LDL, which is also in agreement with previous reports [14–16]. These results confirmed that target TUG1 inhibition might be a promising strategy for therapeutics of atherosclerosis. Previous studies suggested that TUG1 could participate in regulation of multiple diseases by serving as a ceRNA or sponge of miRNAs, such as miR-133a, miR-21, miR-197, miR-26a and miR-374c [12,14–16,36]. However, the mechanism mediated via TUG1 was complex because of the multiple binding sites of miRNAs. To explore the regulatory mechanism, we first confirmed miR-148b as a target of TUG1 using luciferase reporter and RIP assays. The former studies demonstrated the anti-proliferation role of miR-148b in VSMC treated by ox-LDL [18,19]. Our results consistently demonstrated that miR-148b overexpression decreased proliferation and increased apoptosis in VSMC. Moreover, we also found that miR-148b addition weakened the HUVEC apoptosis induced by ox-LDL, which indicated the therapeutic
VSMC was obviously enhanced by TUG1 overexpression, and this effect was counteracted via miR-148b addition (Fig. 7C). Meanwhile, Western blot results also demonstrated that the abundance of IGF2 protein in HUVEC was significantly decreased by silencing TUG1, which was restored by miR-148b absence (Fig. 7D). 4. Discussion LncRNAs play important roles in development, prevention and treatment of atherosclerosis [27]. The former works have indicated that TUG1 is associated with atherosclerosis development [14–16]. This study aimed to explore new mechanism for better understanding the pathogenesis of atherosclerosis. In the present research, we investigated the suppressive role of TUG1 knockdown in atherosclerosis progression and elucidated a novel ceRNA network of TUG1/miR-148b/IGF2 using the cellular model of atherosclerosis. VSMC and HUVEC stimulated by ox-LDL were widely used as model of atherosclerosis in vitro [28–31]. To validate the cellular model of atherosclerosis in our study, ox-LDL was introduced to VSMC and HUVEC. Bcl-2 family is involved in apoptosis with Bcl-2 protein as antiapoptotic marker and Bax as pro-apoptotic marker and the dysfunction of Bcl-2/Bax balance could induce endothelial cell apoptosis, which contributes to atherosclerosis development [32,33]. Moreover, PCNA is a protein associated with cell proliferation, which facilitates DNA 7
Life Sciences 243 (2020) 117287
X. Wu, et al.
effect of miR-148b on atherosclerosis. More importantly, knocking down miR-148b abated the effect of TUG1 silence on VSMC and HUVEC proliferation and apoptosis. This implicated that TUG1 regulated VSMC and HUVEC function by sponging miR-148b. A target is required for the function of miRNAs and here we were the first to validate IGF2 as a target of miR-148b. Sun et al [37] and Wang et al [38] reported that ox-LDL could increase the expression of IGF2 in VSMC and THP-1 macrophages, which implied that IGF2 might contribute to ox-LDL-induced injury. Unsurprisingly, we found that IGF2 restoration abrogated the therapeutic role of miR-148b in atherosclerosis. Furthermore, using the luciferase reporter assay and Western blot we conclude that TUG1 could increase IGF2 expression by competitively sponging miR-148b. However, this study just investigated the role of TUG1 in vitro. The ex vivo and in vivo studies are responsible for elucidating pathogenesis of atherosclerosis [39]. Further ex vivo and in vivo studies will be performed to investigate the regulatory mechanism of TUG1 in atherosclerosis. Considered together, our study demonstrates the therapeutic effect of TUG1 inhibition on atherosclerosis development by attenuating VSMC proliferation and HUVEC apoptosis, possibly by increasing miR148b and decreasing IGF2. This work highlights a new regulatory mechanism for TUG1 in atherosclerosis progression and indicated a novel target for treatment of atherosclerosis. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2020.117287.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Funding
[21]
None.
[22]
Author's contribution
[23]
Xiaoguang Wu and Xiaohui Zheng were the main designers of this study. Jing Cheng, Kai Zhang and Cao Ma performed the experiments and analyzed the data. Xiaohui Zheng drafted the manuscript. All the authors read and approved the final manuscript.
[24]
[25]
Declaration of competing interest [26]
All authors declare no conflicts of interest. [27]
References
[28]
[1] C. Weber, H. Noels, Atherosclerosis: current pathogenesis and therapeutic options, Nat. Med. 17 (11) (2011) 1410–1422, https://doi.org/10.1038/nm.2538. [2] M.R. Bennett, S. Sinha, G.K. Owens, Vascular smooth muscle cells in Atherosclerosis, Circ. Res. 118 (4) (2016) 692–702, https://doi.org/10.1161/ CIRCRESAHA.115.306361. [3] M.A. Gimbrone Jr., G. Garcia-Cardena, Endothelial cell dysfunction and the pathobiology of atherosclerosis, Circ. Res. 118 (4) (2016) 620–636, https://doi.org/ 10.1161/CIRCRESAHA.115.306301. [4] S. Gao, D. Zhao, M. Wang, et al., Association between circulating oxidized LDL and atherosclerotic cardiovascular disease: a meta-analysis of observational studies, Can J Cardiol. 33 (12) (2017) 1624–1632, https://doi.org/10.1016/j.cjca.2017.07.015. [5] C.F. Suciu, M. Prete, P. Ruscitti, et al., Oxidized low density lipoproteins: the bridge between atherosclerosis and autoimmunity. Possible implications in accelerated atherosclerosis and for immune intervention in autoimmune rheumatic disorders, Autoimmun. Rev. 17 (4) (2018) 366–375, https://doi.org/10.1016/j.autrev.2017. 11.028. [6] M. Li, L. Duan, Y. Li, et al., Long noncoding RNA/circular noncoding RNA-miRNAmRNA axes in cardiovascular diseases, Life Sci. 233 (2019) 116440, https://doi. org/10.1016/j.lfs.2019.04.066. [7] Y. Liu, L. Zheng, Q. Wang, et al., Emerging roles and mechanisms of long noncoding RNAs in atherosclerosis, Int. J. Cardiol. 228 (2017) 570–582, https://doi.org/10. 1016/j.ijcard.2016.11.182. [8] K.K. Singh, P.N. Matkar, Y. Pan, et al., Endothelial long non-coding RNAs regulated by oxidized LDL, Mol. Cell. Biochem. 431 (1–2) (2017) 139–149, https://doi.org/ 10.1007/s11010-017-2984-2. [9] A. Leung, K. Stapleton, R. Natarajan, Functional long non-coding RNAs in vascular smooth muscle cells, Curr. Top. Microbiol. Immunol. 394 (2016) 127–141, https:// doi.org/10.1007/82_2015_441. [10] H.J. Sun, B. Hou, X. Wang, et al., Endothelial dysfunction and cardiometabolic
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
8
diseases: role of long non-coding RNAs, Life Sci. 167 (2016) 6–11, https://doi.org/ 10.1016/j.lfs.2016.11.005. S. Ghaforui-Fard, R. Vafaee, M. Taheri, Taurine-upregulated gene 1: a functional long noncoding RNA in tumorigenesis, J. Cell. Physiol. 234 (10) (2019) 17100–17112, https://doi.org/10.1002/jcp.28464. D. Zhao, Z. Liu, H. Zhang, The protective effect of the TUG1/miR197/MAPK1 axis on lipopolysaccharideinduced podocyte injury, Mol. Med. Rep. 20 (1) (2019) 49–56, https://doi.org/10.3892/mmr.2019.10216. Q. Su, Y. Liu, X.W. Lv, et al., Inhibition of lncRNA TUG1 upregulates miR-142-3p to ameliorate myocardial injury during ischemia and reperfusion via targeting HMGB1- and Rac1-induced autophagy, J Mol Cell Cardiol. 133 (2019) 12–25, https://doi.org/10.1016/j.yjmcc.2019.05.021. L. Zhang, H. Cheng, Y. Yue, et al., TUG1 knockdown ameliorates atherosclerosis via up-regulating the expression of miR-133a target gene FGF1, Cardiovasc Pathol. 33 (2018) 6–15, https://doi.org/10.1016/j.carpath.2017.11.004. F.P. Li, D.Q. Lin, L.Y. Gao, LncRNA TUG1 promotes proliferation of vascular smooth muscle cell and atherosclerosis through regulating miRNA-21/PTEN axis, Eur. Rev. Med. Pharmacol. Sci. 22 (21) (2018) 7439–7447, https://doi.org/10.26355/eurrev_ 201811_16284. C. Chen, G. Cheng, X. Yang, et al., Tanshinol suppresses endothelial cells apoptosis in mice with atherosclerosis via lncRNA TUG1 up-regulating the expression of miR26a, Am. J. Transl. Res. 8 (7) (2016) 2981–2991. A. Schober, C. Weber, Mechanisms of MicroRNAs in atherosclerosis, Annu. Rev. Pathol. 11 (2016) 583–616, https://doi.org/10.1146/annurev-pathol-012615044135. L. Zhang, H. Cheng, Y. Yue, et al., H19 knockdown suppresses proliferation and induces apoptosis by regulating miR-148b/WNT/beta-catenin in ox-LDLstimulated vascular smooth muscle cells, J. Biomed. Sci. 25 (1) (2018) 11, https://doi.org/10. 1186/s12929-018-0418-4. X. Zhang, H. Shi, Y. Wang, et al., Down-regulation of hsa-miR-148b inhibits vascular smooth muscle cells proliferation and migration by directly targeting HSP90 in atherosclerosis, Am. J. Transl. Res. 9 (2) (2017) 629–637. L.A. Bach, Endothelial cells and the IGF system, J. Mol. Endocrinol. 54 (1) (2015) R1–13, https://doi.org/10.1530/JME-14-0215. D. Bergman, M. Halje, M. Nordin, et al., Insulin-like growth factor 2 in development and disease: a mini-review, Gerontology. 59 (3) (2013) 240–249, https://doi.org/ 10.1159/000343995. X. Liu, B.D. Ma, S. Liu, et al., Long noncoding RNA LINC00341 promotes the vascular smooth muscle cells proliferation and migration via miR-214/FOXO4 feedback loop, Am. J. Transl. Res. 11 (2019) 1835–1842. C.X. Xu, L. Xu, F.Z. Peng, et al., MiR-647 promotes proliferation and migration of ox-LDL-treated vascular smooth muscle cells through regulating PTEN/PI3K/AKT pathway, Eur Rev Med Pharmacol Sci. 23 (2019) 7110–7119, https://doi.org/10. 26355/eurrev_201908_18756. M. Zhang, L. Jiang, Oxidized low-density lipoprotein decreases VEGFR2 expression in HUVECs and impairs angiogenesis, Exp Ther Med 12 (2016) 3742–3748, https:// doi.org/10.3892/etm.2016.3823. L. Gong, Y. Lei, Y. Liu, et al., Vaccarin prevents ox-LDL-induced HUVEC EndMT, inflammation and apoptosis by suppressing ROS/p38 MAPK signaling, Am. J. Transl. Res. 11 (2019) 2140–2154. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(−delta delta C(T)) method, Methods. 25 (4) (2001) 402–408, https://doi.org/10.1006/meth.2001.1262. Z. Zhang, D. Salisbury, T. Sallam, Long noncoding RNAs in atherosclerosis: JACC review topic of the week, J. Am. Coll. Cardiol. 72 (19) (2018) 2380–2390, https:// doi.org/10.1016/j.jacc.2018.08.2161. X. Liu, B.D. Ma, S. Liu, et al., Long noncoding RNA LINC00341 promotes the vascular smooth muscle cells proliferation and migration via miR-214/FOXO4 feedback loop, Am J Transl Res. 11 (3) (2019) 1835–1842. L. Zhang, C. Zhou, Q. Qin, et al., LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis, J Cell Biochem. 120 (9) (2019) 14670–14678, https://doi.org/10.1002/jcb.28728. X. Xu, C. Ma, Z. Duan, et al., lncRNA ZEB1-AS1 mediates oxidative low-density lipoprotein-mediated endothelial cells injury by post-transcriptional stabilization of NOD2, Front Pharmacol. 10 (2019) 397, https://doi.org/10.3389/fphar.2019. 00397. L. Cao, Z. Zhang, Y. Li, et al., LncRNA H19/miR-let-7 axis participates in the regulation of ox-LDL-induced endothelial cell injury via targeting periostin, Int Immunopharmacol. 72 (2019) 496–503, https://doi.org/10.1016/j.intimp.2019. 04.042. J.C. Choy, D.J. Granville, D.W. Hunt, et al., Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis, J. Mol. Cell. Cardiol. 33 (9) (2001) 1673–1690, https://doi.org/10.1006/jmcc.2001.1419. F. Edlich, BCL-2 proteins and apoptosis: recent insights and unknowns, Biochem. Biophys. Res. Commun. 500 (1) (2018) 26–34, https://doi.org/10.1016/j.bbrc. 2017.06.190. S.C. Wang, PCNA: a silent housekeeper or a potential therapeutic target? Trends Pharmacol. Sci. 35 (4) (2014) 178–186, https://doi.org/10.1016/j.tips.2014.02. 004. W. Leung, R.M. Baxley, G.L. Moldovan, et al., Mechanisms of DNA damage tolerance: post-translational regulation of PCNA, Genes (Basel) 10 (1) (2018) 10, https://doi.org/10.3390/genes10010010. L. Yang, H. Liang, L. Shen, et al., LncRNA Tug1 involves in the pulmonary vascular remodeling in mice with hypoxic pulmonary hypertension via the microRNA-374cmediated Foxc1, Life Sci. 237 (2019) 116769, https://doi.org/10.1016/j.lfs.2019. 116769.
Life Sciences 243 (2020) 117287
X. Wu, et al.
0194-1. [39] F. Hajizadeh-Sharafabad, Z. Ghoreishi, V. Maleki, et al., Mechanistic insights into the effect of lutein on atherosclerosis, vascular dysfunction, and related risk factors: a systematic review of in vivo, ex vivo and in vitro studies, Pharmacol Res. 149 (2019) 104477, https://doi.org/10.1016/j.phrs.2019.104477.
[37] Y. Sun, D. Chen, L. Cao, et al., MiR-490-3p modulates the proliferation of vascular smooth muscle cells induced by ox-LDL through targeting PAPP-A, Cardiovasc. Res. 100 (2) (2013) 272–279, https://doi.org/10.1093/cvr/cvt172. [38] Y.C. Wang, Y.W. Hu, Y.H. Sha, et al., Ox-LDL upregulates IL-6 expression by enhancing NF-kappaB in an IGF2-dependent manner in THP-1 macrophages, Inflammation 38 (6) (2015) 2116–2123, https://doi.org/10.1007/s10753-015-
9
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
LncRNA TUG1 regulates proliferation and apoptosis by regulating miR148b/IGF2 axis in ox-LDL-stimulated VSMC and HUVEC
T
⁎
Xiaoguang Wu, Xiaohui Zheng , Jing Cheng, Kai Zhang, Cao Ma Department of Emergency, Fuwai Central China Cardiovascular Hospital, Central China Fuwai Hospital of Zhengzhou University, Henan Provincial People's Hospital, Zhengzhou, Henan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherosclerosis TUG1 miR-148b IGF2 VSMC HUVEC
Vascular smooth muscle cell (VSMC) accumulation and endothelial cell dysfunction are associated with pathogenesis of atherosclerosis. Long noncoding RNA taurine up-regulated gene 1 (TUG1) has been reported to play an important role in cardiovascular diseases, including atherosclerosis. However, the regulatory mechanism underlying TUG1 in atherosclerosis is far from understood. VSMC and human umbilical vein endothelial cells (HUVEC) stimulated by oxidized low-density lipoprotein (ox-LDL) were used as cellular model of atherosclerosis. Cell proliferation and apoptosis were detected by CCK-8, flow cytometry and Western blot. The expression levels of TUG1, microRNA (miR)-148b and insulin-like growth factor 2 (IGF2) were measured by quantitative real-time polymerase chain reaction or Western blot. The target association among TUG1, miR-148b and IGF2 was determined by luciferase reporter assay and RNA immunoprecipitation. The expression of TUG1 was increased in ox-LDL-treated VSMC and HUVEC. Silence of TUG1 inhibited proliferation and promoted apoptosis in ox-LDLtreated VSMC but induced proliferation promotion and apoptosis inhibition in HUVEC stimulated by ox-LDL. miR-148b was a target of TUG1 and its knockdown reversed the effect of TUG1 silence on proliferation and apoptosis of VSMC and HUVEC challenged by ox-LDL. IGF2 was a target of miR-148b and miR-148b regulated proliferation and apoptosis in ox-LDL-treated VSMC and HUVEC by targeting IGF2. TUG1 promoted IGF2 protein expression by sponging miR-148b. TUG1 knockdown attenuated ox-LDL-induced injury through regulating proliferation and apoptosis of VSMC and HUVEC by miR-148b/IGF2 axis, providing a novel mechanism for pathogenesis of atherosclerosis.
1. Introduction Atherosclerosis is a common cardiovascular disease with high morbidity and mortality [1]. The aberrant proliferation of vascular smooth muscle cells (VSMC) is associated with progression of early atherosclerosis and vascular injury [2]. Moreover, endothelial cell injury is a key contributor for cardiovascular diseases, including atherosclerosis [3]. Oxidized low-density lipoprotein (ox-LDL) accumulation is one of the major risks of atherosclerosis, which contributes to the accelerated atherosclerosis [4,5]. However, the mechanism underlying how ox-LDL regulates proliferation and apoptosis of VSMC and human umbilical vein endothelial cells (HUVEC) remains largely unclear. Long noncoding RNAs (lncRNAs) with over 200 nucleotides are involved in regulation of cardiovascular diseases by competing endogenous RNA (ceRNA) networks of lncRNA/microRNA (miRNA)/ mRNA axis [6]. Moreover, lncRNAs exhibit essential roles in
atherosclerosis by regulating lipid metabolism, inflammatory response, cell proliferation, apoptosis and migration [7]. Meanwhile, lncRNAs are dysregulated by ox-LDL treatment and associated with the function of VSMC and endothelial cells [8–10]. Taurine up-regulated gene 1 (TUG1) is a functional lncRNA having a vital role in human cancers and diseases [11–13]. In addition, TUG1 could promote macrophages growth and inflammatory response in atherosclerosis [14]. Besides, previous studies indicate that TUG1 could promote atherosclerosis by increasing VSMC proliferation and triggering endothelial cell apoptosis [14–16]. However, the mechanism underlying TUG1 in atherosclerosis has not been fully understood. miRNAs which could be sponged by lncRNAs have important roles in regulation of atherosclerosis development [17]. miR-148b has been reported as an inhibitor of atherosclerosis by decreasing VSMC proliferation and migration but increasing apoptosis [18,19]. Furthermore, insulin-like growth factor (IGF) system is responsible for the function of
⁎ Corresponding author at: Department of Emergency, Central China Fuwai Hospital of Zhengzhou University, Fuwai Central China Cardiovascular Hospital, Henan Provincial People's Hospital, No.1 Fuwai Road, Zhengzhou 451464, Henan, China. E-mail address: [email protected] (X. Zheng).
https://doi.org/10.1016/j.lfs.2020.117287 Received 12 October 2019; Received in revised form 30 December 2019; Accepted 7 January 2020 Available online 08 January 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 1. The expression of TUG1 is increased in VSMC and HUVEC stimulated by ox-LDL. (A and B) qRT-PCR was performed to detect the expression of TUG1 in VSMC and HUVEC treated by ox-LDL. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group (non-treated with ox-LDL group).
Fig. 2. Knockdown of TUG1 regulates proliferation and apoptosis in VSMC and HUVEC stimulated by ox-LDL. (A) The abundance of TUG1 was measured in VSMC and HUVEC after transfection of si-TUG1 or si-NC in the presence of ox-LDL or not. Cell viability (B and C), apoptosis (D) and protein levels of Bax, Bcl-2 and PCNA (E) were determined in VSMC and HUVEC transfected with si-TUG1 or si-NC after treatment of ox-LDL or not. **P < 0.01, ***P < 0.001, compared with si-NC group or ox-LDL + si-NC group.
two cell lines. Additionally, we explored the potential regulatory network of TUG1/miR-148b/IGF2 axis.
endothelial cells in vascular diseases [20]. IGF2 is one member of IGF family, which is involved in regulation of cell proliferation, migration and survival and associated with cardiovascular diseases [21]. Intriguingly, starBase database predicted that TUG1 and IGF2 shared the complementary binding sites of miR-148b, which stimulated us to assume the ceRNA network of TUG1/miR-148b/IGF2. In this study, VSMC and HUVEC were stimulated by ox-LDL as previous studies [22–25]. Moreover, we measured the expression of TUG1 and investigated the effect of TUG1 on cell proliferation and apoptosis in the
2. Materials and methods 2.1. Cell culture, ox-LDL treatment and transfection The human VSMC from human umbilical artery and HUVEC were purchased from BeNa Culture Collection (Beijing, China). VSMC and 2
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 3. TUG1 is a decoy of miR-148b. (A) starBase online predicted the complementary sequences between TUG1 and miR-148b. (B and C) Luciferase activity was analyzed in VSMC and HUVEC co-transfected with miR-NC or miR-148b and TUG1-WT or TUG1-MUT. (D and E) RIP assay was performed in VSMC and HUVEC transfected with miR-148 or miR-NC and TUG1 level was detected by qRT-PCR. (F) qRT-PCR was used to measure the level of miR-148b in VSMC and HUVEC after treatment of ox-LDL. (G and H) The expression of miR-148b was examined in VSMC and HUVEC transfected with pcDNA, TUG1, si-NC or si-TUG1. **P < 0.01, ***P < 0.001, compared with miR-NC group for B-E, with control group for F, with pcDNA or si-NC group for G and H.
2.2. CCK-8
HUVEC were cultured in DMEM (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum and antibiotics and maintained at 37 °C with 5% CO2. For construct of atherosclerosis model in vitro, VSMC and HUVEC were stimulated with 100 μg/ml ox-LDL (Solarbio, Beijing, China) [22–25]. The overexpression vectors of TUG1 and IGF2 were generated by inserting into pcDNA3.1 empty vector (pcDNA) (Thermo Fisher Scientific, Wilmington, DE, USA). siRNA against TUG1 (si-TUG1) ( 5′-GGATATAGCCAGAGAACAA-3′), siRNA negative control (si-NC) ( 5′-GTTCTCCGAACGTGTCACGT-3′), miR-148b mimic (miR-148b) ( 5′-UCAGUGCAUCACAGAACUUUGU-3′), miRNA negative control (miRNC) (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-148b inhibitor (antimiR-148b) (5′-ACAAAGUUCUGUGAUGCACUGA-3′) and inhibitor negative control (anti-miR-NC) (5′-CAGUACUUUUGUGUAGUACAA-3′) were generated by GenePharm (Shanghai, China). VSMC and HUVEC were transfected with constructed oligonucleotides at 30 nM concentration using Lipofectamine 2000 (Thermo Fisher Scientific) for 24 h. Subsequently, transfected cells were collected for further experiments.
Transfected or non-transfected VSMC and HUVEC (3000 cells/well) were seeded into 96-well plates in triplicate and then incubated with ox-LDL. After 0, 24, 48 and 72 h, 10 μl CCK-8 solution (Beyotime, Shanghai, China) was added into each well and cells were continuously cultured for 3 h. The optical density value of each well at 450 nm was measured by microplate reader (Bio-Rad, Hercules, CA, USA). 2.3. Flow cytometry VSMC and HUVEC were stimulated with ox-LDL for 72 h. The cells were collected and incubated in binding buffer, followed by staining with Annexin V-FITC and PI solution in Annexin V-FITC Apoptosis Detection kit (Beyotime). The apoptotic cells were measured by a flow cytometer and the apoptotic rate of VSMC and HUVEC at early and late apoptosis was presented as the percentage of cells with Annexin V-FITC staining positive and PI staining negative or positive. 2.4. Western blot ProteoPrep® Total Extraction Sample Kit and Bicinchoninic Acid Kit 3
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 4. Inhibition of miR-148b reverses the effect of TUG1 knockdown on proliferation and apoptosis in VSMC and HUVEC stimulated by ox-LDL. (A and B) The expression of miR-148b was detected in VSMC and HUVEC after transfection of si-NC, si-TUG1, si-TUG1 + anti-miR-NC or anti-miR-148b. Cell viability (C and D), apoptosis (E and F) and protein abundances of Bax, Bcl-2 and PCNA (G and H) were determined in VSMC and HUVEC transfected with si-NC, si-TUG1, siTUG1 + anti-miR-NC or anti-miR-148b after treatment of ox-LDL. **P < 0.01, ***P < 0.001, compared with si-NC or si-TUG1 + anti-miR-NC group.
TUG1, IGF2 and miR-148b were analyzed by 2−ΔΔCt method [26].
(Sigma) were used for extraction and concentration determination of total protein from VSMC and HUVEC. The protein lysates (30 μg) were separated by SDS-PAGE and then electro-transferred to PVDF membranes (Millipore, Billerica, MA, USA). Following the block using 5% non-fat milk, the membranes were incubated with primary antibodies and secondary antibody, including anti-Bax (ab77566, 1:1000 dilution, 21 kDa), anti-Bcl-2 (ab201566, 1:2000 dilution, 26 kDa), anti-PCNA (ab220208, 1:2000 dilution, 29 kDa), anti-IGF2 (ab63294, 1:1000 dilution, 50 kDa) and HRP-conjugated IgG (ab6728, 1:8000 dilution), which were purchased from Abcam (Cambridge, MA, USA). Moreover, anti-β-actin (ab49900, 1:30,000 dilution, 42 kDa, Abcam) was used as loading control. The protein blot signaling was visualized using enhanced chemiluminescence solution (Beyotime) and film in the dark.
2.6. Luciferase reporter assay and RNA immunoprecipitation (RIP) The potential complementary sequences of miR-148b and TUG1 or IGF2 were predicted by starBase online (http://starbase.sysu.edu.cn/). The wild-type luciferase reporter vectors of TUG1 and IGF2 (TUG1-WT and IGF2-WT) were generated by inserting the sequences of TUG1 or IGF2 containing miR-148b binding sites into pmirGLO luciferase reporter vector (Promega, Madison, WI, USA). Their corresponding mutants (TUG1-MUT and IGF2-MUT) were obtained by mutating the seed sites of miR-148b. VSMC and HUVEC were transfected with TUG1-WT, TUG1-MUT, IGF2-WT or IGF2-MUT, along with miR-148b or miR-NC. The luciferase reporter assay was carried out using luciferase activity analysis kit (Promega) after the transfection for 24 h. RIP assay was performed in VSMC and HUVEC transfected with miR-148b or miR-NC using Magna RNA immunoprecipitation kit (Millipore) in accordance with the manufacturer's protocols. In brief, the lysed cells were incubated with RIP buffer with magnetic beads conjugated by anti-Ago2 (ab32381, Abcam) or IgG (AP112, Sigma). The enriched abundances of TUG1 and IGF2 by RIP were determined by qRT-PCR.
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) A total of 1 μg RNA isolated using Trizol reagent (Thermo Fisher Scientific) was reversely transcribed to cDNA with the special RT-PCR Kit (Thermo Fisher Scientific) following the manufacturer's instructions. The synthesized cDNA was diluted by 1:5 and then qRT-PCR was performed on ABI 7500 Real-time PCR system (Bio-Rad) with SYBR Green mix (Thermo Fisher Scientific) and primers: TUG1 (Forward, 5′-TAGC AGTTCCCCAATCCTTG-3′; Reverse, 5′-CACAAATTCCCATCATTC CC3′); IGF2 (Forward, 5′-AGACCCTTTGCGGTGGAGA-3′; Reverse, 5′-GG AAACATCTCGCTCGGACT-3′); miR-148b(Forward, 5′-ACACTCCAGCT GGGT CAGTGCATC-3′; Reverse, 5′-CTCAACTGGTGTCGTGGA-3′). GAPDH (Forward, 5′-CAGCCTCAAGATCATCAGCA-3′; Reverse, 5′-TGT GGTCATGAGTCCTT CCA-3′) and U6 (Forward, 5′-CGCTTCGGCAGCA CATATAC-3′; Reverse, 5′-TTCACGAATTTGCGTGTCAT-3′) were used as internal control for RNA expression. The relative expression levels of
2.7. Statistical analysis The experiments were repeated three times with three replicates. The data were presented as mean ± standard deviation and analyzed by GraphPad Prism 7.0. The difference between two or more groups was compared by Student's t-test or ANOVA with Tukey's post hoc test. It was regarded as statistically significant when P < 0.05 (*P < 0.05, 4
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 5. IGF2 is a target of miR-148b. (A) The binding sites of miR-148b and IGF2 were provided by starBase online. (B-E) Luciferase reporter and RIP assays were performed in VSMC and HUVEC transfected with miR-148b or miR-NC to validate the association between miR-148b and IGF2. (F) The protein level of IGF2 was measured in VSMC and HUVEC after stimulation of ox-LDL by Western blot. (G and H) Western blot assay was carried out to detect the level of IGF2 protein in VSMC and HUVEC transfected with miR-NC, miR-148b, anti-miR-NC or anti-miR-148b. **P < 0.01, ***P < 0.001, compared with miR-NC group for B-E, with control group for F, with miR-NC or anti-miR-NC group for G and H.
decreased viability of VSMC and increased viability of HUVEC in the presence of ox-LDL at 48 and 72 h (Fig. 2B and C). Additionally, the analysis of flow cytometry described that down-regulation of TUG1 notably enhanced apoptosis in VSMC but reduced the apoptosis in HUVEC stimulated by ox-LDL (Fig. 2D). What's more, Bax protein level was increased but Bcl-2 and PCNA protein abundances were decreased in VSMC treated by ox-LDL, while these events were opposite in oxLDL-treated HUVEC (Fig. 2E).
**P < 0.01, ***P < 0.001). 3. Results 3.1. TUG1 expression is enhanced in ox-LDL-treated VSMC and HUVEC The cellular model of atherosclerosis was constructed using VSMC and HUVEC stimulated by ox-LDL. As shown in Supplementary Fig. 1A–D, treatment of ox-LDL promoted VSMC viability and inhibited apoptosis, while it decreased viability and increased apoptosis in HUVECs. Moreover, the expression level of TUG1 was measured in VSMC and HUVEC treated via different concentrations of ox-LDL. Results showed that TUG1 expression was evidently elevated in VSMC and HUVEC after stimulation of ox-LDL in a concentration dependent manner (Fig. 1A and B). 100 μg/ml ox-LDL that induced extremely significant up-regulation of TUG1 (P < 0.001) was used for further experiments.
3.3. TUG1 is a decoy of miR-148b To explore the underlying mechanism addressed by TUG1, starBase database was used to determine the putative complementary sequences between TUG1 and miR-148b at position: chr22: 31372748–31372770 (Fig. 3A). To validate their association, the constructed luciferase reporter vectors TUG1-WT or TUG1-MUT were transfected into VSMC and HUVEC. The luciferase reporter assay displayed that miR-148b addition led to great loss of luciferase activity in VSMC and HUVEC transfected with TUG1-WT, whereas it showed little effect in TUG1MUT group (Fig. 3B and C). Moreover, overexpression of miR-148b induced higher enrichment level of TUG1 by Ago2 RIP in VSMC and HUVEC (Fig. 3D and E). In ox-LDL-challenged cells, the expression of miR-148b was specially decreased in comparison to control group (Fig. 3F). Additionally, the abundance of miR-148b in VSMC and
3.2. Silence of TUG1 regulates proliferation and apoptosis in ox-LDLtreated VSMC and HUVEC To investigate the role of TUG1 in atherosclerosis, its abundance was knocked down in VSMC and HUVEC by using si-TUG1, which was confirmed in Fig. 2A. Furthermore, interference of TUG1 obviously 5
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 6. IGF2 attenuates the effect of miR-148b on proliferation and apoptosis in VSMC and HUVEC stimulated by ox-LDL. (A and B) Western blot was conducted to determine the protein level of IGF2 in VSMC and HUVEC after transfection of miR-NC, miR-148b, pcDNA, IGF2, miR-148b + pcDNA or IGF2. Cell viability (C and D), apoptosis (E and F) and protein levels of Bax, Bcl-2 and PCNA (G and H) were measured in VSMC and HUVEC transfected with miR-NC, miR-148b, miR-148b and pcDNA or IGF2 after treatment of ox-LDL. **P < 0.01, ***P < 0.001, compared with miR-NC, pcDNA or miR-148b + pcDNA group.
VSMC and HUVEC after treatment of ox-LDL compared with that in control group (Fig. 5F). Besides, IGF2 protein level was significantly reduced via miR-148b addition and increased by miR-148b deletion in the cells (Fig. 5G and H).
HUVEC was markedly decreased by overexpressing TUG1 and increased by silencing TUG1 (Fig. 3G and H). 3.4. TUG1 regulates proliferation and apoptosis by sponging miR-148b in ox-LDL-treated VSMC and HUVEC
3.6. miR-148b regulates proliferation and apoptosis by targeting IGF2 in oxLDL-treated VSMC and HUVEC
To explore whether TUG1-mediated regulation of atherosclerosis progression was regulated by miR-148b, VSMC and HUVEC were transfected with si-NC, si-TUG1, si-TUG1 and anti-miR-NC or anti-miR148b. As displayed in Fig. 4A and B, the abundance of miR-148b promoted by TUG1 silence was decreased by transfection of anti-miR-148b in VSMC and HUVEC. Moreover, deficiency of miR-148b reversed silencing TUG1-induced viability inhibition in VSMC and viability promotion in HUVEC treated with ox-LDL (Fig. 4C and D). Meanwhile, inhibition of miR-148b weakened the cell apoptosis induced by TUG1 knockdown in ox-LDL-treated VSMC and attenuated the apoptosis suppression caused via TUG1 silence in ox-LDL-stimulated HUVEC (Fig. 4E and F). Furthermore, the effect of TUG1 interference on protein levels of Bax, Bcl-2 and PCNA in VSMC and HUVEC challenged by oxLDL was abolished via miR-148b exhaustion (Fig. 4G and H).
To explore whether IGF2 was involved in miR-148b-mediated regulatory mechanism, VSMC and HUVEC were transfected with miR-NC, miR-148b, pcDNA, IGF2, miR-148b + pcDNA or IGF2. After the transfection, the protein level of IGF2 was inhibited by miR-148b addition, and restored by introduction of IGF2 in VSMC and HUVEC (Fig. 6A and B). Furthermore, overexpression of miR-148b significantly repressed the viability of VSMC treated by ox-LDL and increased the viability of HUVEC stimulated by ox-LDL at 72 h (Fig. 6C and D). Additionally, addition of miR-148b remarkably promoted or suppressed apoptosis in VSMC or HUVEC treated by ox-LDL, respectively (Fig. 6E and F). miR-148b overexpression increased Bax protein and reduced Bcl-2 and PCNA protein levels in VSMC treated by ox-LDL and reduced Bax level and increased Bcl-2 and PCNA protein levels in ox-LDLchallenged HUVEC (Fig. 6G and H). However, IGF2 overexpression led to opposite effect on these events and weakened the role of miR-148 (Fig. 6C–H).
3.5. IGF2 is a target of miR-148b In order to further elucidate the regulatory mechanism, the target of miR-148b was searched by starBase database, which predicted the binding sites of miR-148b and IGF2 at position: chr11:2151600–2151605 (Fig. 5A). Moreover, luciferase reporter assay was used to validate this prediction and results showed that in IGF2-WT group, transfection of miR-148b resulted in obvious reduction of luciferase activity in VSMC and HUVEC, while it did not affect the activity when mutating the seed sites in IGF2-MUT group (Fig. 5B and C). Meanwhile, the level of IGF2 enriched by Ago2 RIP was higher in the cells transfected with miR-148b than that in miR-NC group (Fig. 5D and E). Furthermore, the protein level of IGF2 was markedly enhanced in
3.7. TUG1 regulates IGF2 expression by sponging miR-148b To validate the interaction between TUG1 and IGF2, the luciferase reporter assay was performed. As shown in Fig. 7A and B, the luciferase activity was greatly decreased by overexpression of miR-148a in VSMC and HUVEC transfected with IGF2-WT luciferase reporter vector. In addition, introduction of TUG1 evidently increased the luciferase activity and abolished the suppressive role of miR-148b. which was abated by introduction of TUG1. Moreover, the protein level of IGF2 in 6
Life Sciences 243 (2020) 117287
X. Wu, et al.
Fig. 7. TUG1 regulates IGF2 expression by targeting miR-148b. (A and B) Luciferase activity was determined in VSMC and HUVEC transfected with IGF2-WT, IGF2-WT + miR-148b, IGF2-WT + TUG1, IGF2-WT + miR-148b + pcDNA or IGF2WT + miR-148b + TUG1. (C) Western blot was conducted to detect the protein level of IGF2 in VSMC transfected with pcDNA, TUG1, TUG1 and miR-NC or miR-148b. (D) The protein level of IGF2 was measured in HUVEC transfected with si-NC, siTUG1, si-TUG1 and anti-miR-NC or anti-miR-148b. **P < 0.01, ***P < 0.001, compared with IGF2-WT or IGF2-WT + miR-148b + pcDNA group for A and B, with pcDNA or TUG1 + miR-NC group for C, with si-NC or si-TUG1 + anti-miR-NC group for D.
damage tolerance and attenuates apoptosis [34,35]. By measurement of cell viability, apoptosis and protein levels of Bax, Bcl-2 and PCNA, VSMC proliferation and HUVEC apoptosis were induced by ox-LDL treatment, indicating that the cellular model of atherosclerosis was corroborated. Moreover, we found that ox-LDL exposure increased TUG1 abundance in the cells, which reflected that high expression of TUG1 might be a contributor of atherosclerosis development. Furthermore, loss-of-function experiments revealed that TUG1 knockdown attenuated VSMC proliferation and HUVEC apoptosis induced via ox-LDL, which is also in agreement with previous reports [14–16]. These results confirmed that target TUG1 inhibition might be a promising strategy for therapeutics of atherosclerosis. Previous studies suggested that TUG1 could participate in regulation of multiple diseases by serving as a ceRNA or sponge of miRNAs, such as miR-133a, miR-21, miR-197, miR-26a and miR-374c [12,14–16,36]. However, the mechanism mediated via TUG1 was complex because of the multiple binding sites of miRNAs. To explore the regulatory mechanism, we first confirmed miR-148b as a target of TUG1 using luciferase reporter and RIP assays. The former studies demonstrated the anti-proliferation role of miR-148b in VSMC treated by ox-LDL [18,19]. Our results consistently demonstrated that miR-148b overexpression decreased proliferation and increased apoptosis in VSMC. Moreover, we also found that miR-148b addition weakened the HUVEC apoptosis induced by ox-LDL, which indicated the therapeutic
VSMC was obviously enhanced by TUG1 overexpression, and this effect was counteracted via miR-148b addition (Fig. 7C). Meanwhile, Western blot results also demonstrated that the abundance of IGF2 protein in HUVEC was significantly decreased by silencing TUG1, which was restored by miR-148b absence (Fig. 7D). 4. Discussion LncRNAs play important roles in development, prevention and treatment of atherosclerosis [27]. The former works have indicated that TUG1 is associated with atherosclerosis development [14–16]. This study aimed to explore new mechanism for better understanding the pathogenesis of atherosclerosis. In the present research, we investigated the suppressive role of TUG1 knockdown in atherosclerosis progression and elucidated a novel ceRNA network of TUG1/miR-148b/IGF2 using the cellular model of atherosclerosis. VSMC and HUVEC stimulated by ox-LDL were widely used as model of atherosclerosis in vitro [28–31]. To validate the cellular model of atherosclerosis in our study, ox-LDL was introduced to VSMC and HUVEC. Bcl-2 family is involved in apoptosis with Bcl-2 protein as antiapoptotic marker and Bax as pro-apoptotic marker and the dysfunction of Bcl-2/Bax balance could induce endothelial cell apoptosis, which contributes to atherosclerosis development [32,33]. Moreover, PCNA is a protein associated with cell proliferation, which facilitates DNA 7
Life Sciences 243 (2020) 117287
X. Wu, et al.
effect of miR-148b on atherosclerosis. More importantly, knocking down miR-148b abated the effect of TUG1 silence on VSMC and HUVEC proliferation and apoptosis. This implicated that TUG1 regulated VSMC and HUVEC function by sponging miR-148b. A target is required for the function of miRNAs and here we were the first to validate IGF2 as a target of miR-148b. Sun et al [37] and Wang et al [38] reported that ox-LDL could increase the expression of IGF2 in VSMC and THP-1 macrophages, which implied that IGF2 might contribute to ox-LDL-induced injury. Unsurprisingly, we found that IGF2 restoration abrogated the therapeutic role of miR-148b in atherosclerosis. Furthermore, using the luciferase reporter assay and Western blot we conclude that TUG1 could increase IGF2 expression by competitively sponging miR-148b. However, this study just investigated the role of TUG1 in vitro. The ex vivo and in vivo studies are responsible for elucidating pathogenesis of atherosclerosis [39]. Further ex vivo and in vivo studies will be performed to investigate the regulatory mechanism of TUG1 in atherosclerosis. Considered together, our study demonstrates the therapeutic effect of TUG1 inhibition on atherosclerosis development by attenuating VSMC proliferation and HUVEC apoptosis, possibly by increasing miR148b and decreasing IGF2. This work highlights a new regulatory mechanism for TUG1 in atherosclerosis progression and indicated a novel target for treatment of atherosclerosis. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2020.117287.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Funding
[21]
None.
[22]
Author's contribution
[23]
Xiaoguang Wu and Xiaohui Zheng were the main designers of this study. Jing Cheng, Kai Zhang and Cao Ma performed the experiments and analyzed the data. Xiaohui Zheng drafted the manuscript. All the authors read and approved the final manuscript.
[24]
[25]
Declaration of competing interest [26]
All authors declare no conflicts of interest. [27]
References
[28]
[1] C. Weber, H. Noels, Atherosclerosis: current pathogenesis and therapeutic options, Nat. Med. 17 (11) (2011) 1410–1422, https://doi.org/10.1038/nm.2538. [2] M.R. Bennett, S. Sinha, G.K. Owens, Vascular smooth muscle cells in Atherosclerosis, Circ. Res. 118 (4) (2016) 692–702, https://doi.org/10.1161/ CIRCRESAHA.115.306361. [3] M.A. Gimbrone Jr., G. Garcia-Cardena, Endothelial cell dysfunction and the pathobiology of atherosclerosis, Circ. Res. 118 (4) (2016) 620–636, https://doi.org/ 10.1161/CIRCRESAHA.115.306301. [4] S. Gao, D. Zhao, M. Wang, et al., Association between circulating oxidized LDL and atherosclerotic cardiovascular disease: a meta-analysis of observational studies, Can J Cardiol. 33 (12) (2017) 1624–1632, https://doi.org/10.1016/j.cjca.2017.07.015. [5] C.F. Suciu, M. Prete, P. Ruscitti, et al., Oxidized low density lipoproteins: the bridge between atherosclerosis and autoimmunity. Possible implications in accelerated atherosclerosis and for immune intervention in autoimmune rheumatic disorders, Autoimmun. Rev. 17 (4) (2018) 366–375, https://doi.org/10.1016/j.autrev.2017. 11.028. [6] M. Li, L. Duan, Y. Li, et al., Long noncoding RNA/circular noncoding RNA-miRNAmRNA axes in cardiovascular diseases, Life Sci. 233 (2019) 116440, https://doi. org/10.1016/j.lfs.2019.04.066. [7] Y. Liu, L. Zheng, Q. Wang, et al., Emerging roles and mechanisms of long noncoding RNAs in atherosclerosis, Int. J. Cardiol. 228 (2017) 570–582, https://doi.org/10. 1016/j.ijcard.2016.11.182. [8] K.K. Singh, P.N. Matkar, Y. Pan, et al., Endothelial long non-coding RNAs regulated by oxidized LDL, Mol. Cell. Biochem. 431 (1–2) (2017) 139–149, https://doi.org/ 10.1007/s11010-017-2984-2. [9] A. Leung, K. Stapleton, R. Natarajan, Functional long non-coding RNAs in vascular smooth muscle cells, Curr. Top. Microbiol. Immunol. 394 (2016) 127–141, https:// doi.org/10.1007/82_2015_441. [10] H.J. Sun, B. Hou, X. Wang, et al., Endothelial dysfunction and cardiometabolic
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
8
diseases: role of long non-coding RNAs, Life Sci. 167 (2016) 6–11, https://doi.org/ 10.1016/j.lfs.2016.11.005. S. Ghaforui-Fard, R. Vafaee, M. Taheri, Taurine-upregulated gene 1: a functional long noncoding RNA in tumorigenesis, J. Cell. Physiol. 234 (10) (2019) 17100–17112, https://doi.org/10.1002/jcp.28464. D. Zhao, Z. Liu, H. Zhang, The protective effect of the TUG1/miR197/MAPK1 axis on lipopolysaccharideinduced podocyte injury, Mol. Med. Rep. 20 (1) (2019) 49–56, https://doi.org/10.3892/mmr.2019.10216. Q. Su, Y. Liu, X.W. Lv, et al., Inhibition of lncRNA TUG1 upregulates miR-142-3p to ameliorate myocardial injury during ischemia and reperfusion via targeting HMGB1- and Rac1-induced autophagy, J Mol Cell Cardiol. 133 (2019) 12–25, https://doi.org/10.1016/j.yjmcc.2019.05.021. L. Zhang, H. Cheng, Y. Yue, et al., TUG1 knockdown ameliorates atherosclerosis via up-regulating the expression of miR-133a target gene FGF1, Cardiovasc Pathol. 33 (2018) 6–15, https://doi.org/10.1016/j.carpath.2017.11.004. F.P. Li, D.Q. Lin, L.Y. Gao, LncRNA TUG1 promotes proliferation of vascular smooth muscle cell and atherosclerosis through regulating miRNA-21/PTEN axis, Eur. Rev. Med. Pharmacol. Sci. 22 (21) (2018) 7439–7447, https://doi.org/10.26355/eurrev_ 201811_16284. C. Chen, G. Cheng, X. Yang, et al., Tanshinol suppresses endothelial cells apoptosis in mice with atherosclerosis via lncRNA TUG1 up-regulating the expression of miR26a, Am. J. Transl. Res. 8 (7) (2016) 2981–2991. A. Schober, C. Weber, Mechanisms of MicroRNAs in atherosclerosis, Annu. Rev. Pathol. 11 (2016) 583–616, https://doi.org/10.1146/annurev-pathol-012615044135. L. Zhang, H. Cheng, Y. Yue, et al., H19 knockdown suppresses proliferation and induces apoptosis by regulating miR-148b/WNT/beta-catenin in ox-LDLstimulated vascular smooth muscle cells, J. Biomed. Sci. 25 (1) (2018) 11, https://doi.org/10. 1186/s12929-018-0418-4. X. Zhang, H. Shi, Y. Wang, et al., Down-regulation of hsa-miR-148b inhibits vascular smooth muscle cells proliferation and migration by directly targeting HSP90 in atherosclerosis, Am. J. Transl. Res. 9 (2) (2017) 629–637. L.A. Bach, Endothelial cells and the IGF system, J. Mol. Endocrinol. 54 (1) (2015) R1–13, https://doi.org/10.1530/JME-14-0215. D. Bergman, M. Halje, M. Nordin, et al., Insulin-like growth factor 2 in development and disease: a mini-review, Gerontology. 59 (3) (2013) 240–249, https://doi.org/ 10.1159/000343995. X. Liu, B.D. Ma, S. Liu, et al., Long noncoding RNA LINC00341 promotes the vascular smooth muscle cells proliferation and migration via miR-214/FOXO4 feedback loop, Am. J. Transl. Res. 11 (2019) 1835–1842. C.X. Xu, L. Xu, F.Z. Peng, et al., MiR-647 promotes proliferation and migration of ox-LDL-treated vascular smooth muscle cells through regulating PTEN/PI3K/AKT pathway, Eur Rev Med Pharmacol Sci. 23 (2019) 7110–7119, https://doi.org/10. 26355/eurrev_201908_18756. M. Zhang, L. Jiang, Oxidized low-density lipoprotein decreases VEGFR2 expression in HUVECs and impairs angiogenesis, Exp Ther Med 12 (2016) 3742–3748, https:// doi.org/10.3892/etm.2016.3823. L. Gong, Y. Lei, Y. Liu, et al., Vaccarin prevents ox-LDL-induced HUVEC EndMT, inflammation and apoptosis by suppressing ROS/p38 MAPK signaling, Am. J. Transl. Res. 11 (2019) 2140–2154. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(−delta delta C(T)) method, Methods. 25 (4) (2001) 402–408, https://doi.org/10.1006/meth.2001.1262. Z. Zhang, D. Salisbury, T. Sallam, Long noncoding RNAs in atherosclerosis: JACC review topic of the week, J. Am. Coll. Cardiol. 72 (19) (2018) 2380–2390, https:// doi.org/10.1016/j.jacc.2018.08.2161. X. Liu, B.D. Ma, S. Liu, et al., Long noncoding RNA LINC00341 promotes the vascular smooth muscle cells proliferation and migration via miR-214/FOXO4 feedback loop, Am J Transl Res. 11 (3) (2019) 1835–1842. L. Zhang, C. Zhou, Q. Qin, et al., LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis, J Cell Biochem. 120 (9) (2019) 14670–14678, https://doi.org/10.1002/jcb.28728. X. Xu, C. Ma, Z. Duan, et al., lncRNA ZEB1-AS1 mediates oxidative low-density lipoprotein-mediated endothelial cells injury by post-transcriptional stabilization of NOD2, Front Pharmacol. 10 (2019) 397, https://doi.org/10.3389/fphar.2019. 00397. L. Cao, Z. Zhang, Y. Li, et al., LncRNA H19/miR-let-7 axis participates in the regulation of ox-LDL-induced endothelial cell injury via targeting periostin, Int Immunopharmacol. 72 (2019) 496–503, https://doi.org/10.1016/j.intimp.2019. 04.042. J.C. Choy, D.J. Granville, D.W. Hunt, et al., Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis, J. Mol. Cell. Cardiol. 33 (9) (2001) 1673–1690, https://doi.org/10.1006/jmcc.2001.1419. F. Edlich, BCL-2 proteins and apoptosis: recent insights and unknowns, Biochem. Biophys. Res. Commun. 500 (1) (2018) 26–34, https://doi.org/10.1016/j.bbrc. 2017.06.190. S.C. Wang, PCNA: a silent housekeeper or a potential therapeutic target? Trends Pharmacol. Sci. 35 (4) (2014) 178–186, https://doi.org/10.1016/j.tips.2014.02. 004. W. Leung, R.M. Baxley, G.L. Moldovan, et al., Mechanisms of DNA damage tolerance: post-translational regulation of PCNA, Genes (Basel) 10 (1) (2018) 10, https://doi.org/10.3390/genes10010010. L. Yang, H. Liang, L. Shen, et al., LncRNA Tug1 involves in the pulmonary vascular remodeling in mice with hypoxic pulmonary hypertension via the microRNA-374cmediated Foxc1, Life Sci. 237 (2019) 116769, https://doi.org/10.1016/j.lfs.2019. 116769.
Life Sciences 243 (2020) 117287
X. Wu, et al.
0194-1. [39] F. Hajizadeh-Sharafabad, Z. Ghoreishi, V. Maleki, et al., Mechanistic insights into the effect of lutein on atherosclerosis, vascular dysfunction, and related risk factors: a systematic review of in vivo, ex vivo and in vitro studies, Pharmacol Res. 149 (2019) 104477, https://doi.org/10.1016/j.phrs.2019.104477.
[37] Y. Sun, D. Chen, L. Cao, et al., MiR-490-3p modulates the proliferation of vascular smooth muscle cells induced by ox-LDL through targeting PAPP-A, Cardiovasc. Res. 100 (2) (2013) 272–279, https://doi.org/10.1093/cvr/cvt172. [38] Y.C. Wang, Y.W. Hu, Y.H. Sha, et al., Ox-LDL upregulates IL-6 expression by enhancing NF-kappaB in an IGF2-dependent manner in THP-1 macrophages, Inflammation 38 (6) (2015) 2116–2123, https://doi.org/10.1007/s10753-015-
9