1 Introduction
Atherosclerosis is a complex multifactorial disease characterized by vascular remodeling. Dysfunction of the endothelial lining of lesion-prone areas of the arterial vasculature emerges during the progression of atherosclerosis, including lesion formation, plaque rupture, and thrombosis [
1]. Autophagy exerts a protective mechanism that allows the recycling of defective organelles and proteins to maintain normal cellular homeostasis [
2,
3]. Enhanced autophagy could promote plaque stability and thus serve as a potential anti-atherosclerotic mechanism [
4]. In particular, specific endothelial autophagy deficiency promotes atherosclerotic lesion formation; by contrast, stimulation of endothelial autophagic flux contributes to the prevention of atherogenesis [
5,
6].
Long noncoding RNAs (lncRNAs) are involved in various biological functions [
7]. They play a crucial role in regulating cellular autophagy [
8]. The lncRNA growth arrest-specific 5 (lncR-GAS5) is a multifunctional noncoding RNA [
9]. LncR-GAS5 expression was elevated in the plasma of patients with atherosclerosis and oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs) [
10]. Recent studies demonstrated the involvement of lncR-GAS5 in modulating autophagic flux [
10–
12]. Downregulated lncR-GAS5 restores autophagy in ox-LDL-treated endothelial cells (ECs) [
10]. However, the overexpression of lncR-GAS5 represses excessive autophagy [
12]. Although lncR-GAS5 plays a role in regulating autophagic homeostasis, its specific effects and molecular mechanism on atherogenesis are still not fully elucidated.
lncRNAs could interact with miRNAs. Our previous studies have revealed that miR-26a could attenuate atherogenesis by inhibiting EC apoptosis [
13]. Thus, this study explored the potential miRNAs involved in the anti-atherogenesis effect of lncR-GAS5. We predicted and validated that lncR-GAS5 could bind with miR-193-3p. MiR-193-3p has a protective effect against myocardial damage [
14]. The inhibition of miR-193-3p could inhibit temozolomide-induced autophagy [
15].
This study revealed that lncR-GAS5 overexpression accelerated atherogenesis partially by repressing autophagic flux in the endothelium. Mechanistically, lncR-GAS5 regulates miR-193-5p, thereby inhibiting SRSF10 (a direct target of miR-193-5p). Thus, the lncR-GAS5/miR-193-5p/SRSF10 axis could be a novel therapeutic target for atherosclerosis.
2 Materials and methods
2.1 Mouse model construction
Male ApoE−/− mice were purchased from Nanjing Junke Biological Engineering Co., Ltd., and housed under standard housing conditions at a temperature of 23 ± 1 °C and humidity of 55%–60%. Eight-week-old male mice were randomly divided into the following groups: chow diet (CD), high-fat diet (HFD), HFD + Lv null (Lv-lentiviral empty vectors), HFD + Lv-GAS5 (lentiviral (Lv-GAS5) vectors), HFD + Lv-miR-193-5p negative control (NC), and HFD + Lv-miR-193-5p. The mice were injected with 200 µL of lentivirus via the tail vein and fed with HFD containing 0.3% cholesterol and 21% (wt/wt) fat for 12 weeks. Twelve weeks after treatment, the mice (20 weeks old) were sacrificed for aortic lesion size and biochemical measurements. All procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University (Protocol (2009)-11). The use of animals was compliant with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).
2.2 Lipid and lipoprotein measurements
Mice were fasted for 12–14 h, and the blood samples were collected by retro-orbital venous plexus puncture. Next, plasma was separated by centrifugation and stored at −80 °C. Total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were enzymatically detected (Nanjing Jiancheng Biology Engineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s instructions.
2.3 En face Oil Red O (ORO) staining
ORO stock solution (35 mL; 0.2% weight/volume in methanol) was added into 10 mL of 1 M NaOH and filtered. Aortas were rinsed with 78% methanol, stained with 0.16% ORO solution for 50 min, and then rinsed in 78% methanol for 5 min. Finally, the lesion area was assessed as a percentage of the ORO-stained area in the total aorta area.
2.4 Plaque analysis
The hearts were fixed O/N in 4% PFA, dehydrated with 30% sucrose O/N, and then embedded in OCT and frozen at −80 °C. For morphometric analysis, 6-μm-thick serial sections of aortic root were cut using a cryostat. Hematoxylin and eosin (H&E) staining was performed for the quantification of lesion area. Aortic root lesion size was obtained by averaging the lesion areas. The lipid and collagen contents of aortic root lesion were confirmed by using ORO and Masson staining kits (Nanjing Jiancheng Biology Engineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s instructions, respectively.
2.5 Cell culture and transfection
HAECs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in Endothelial Cell Medium supplemented with growth factors, 5% FBS, and 1% penicillin/streptomycin. HAECs were transiently transfected with lncR-GAS5 siRNA (si-GAS5) and NC or its plasmid encoded with lncR-GAS5 sequence (GAS5-P) and negative controls (NC-P), miR-193-5p mimics (miR-193-5p mi), miR-193-5p inhibitor (miR-193-5p inh), and SRSF10 siRNA (si-SRSF10) or NC (RiboBio Co., Ltd., Guangzhou, Guangdong, China), using Lipofectamine 2000 reagent (Invitrogen, CA, Carlsbad, USA). Twenty-four hours after transfection, the medium was replaced with or without 3-MA (5 mM) or rapamycin (100 nM) for 4 h, respectively. Next, the cells were collected for the following experiments.
2.6 Immunofluorescence
Immunofluorescence staining was performed as previously described [
16]. Briefly, the aorta root and HAECs were fixed with 4% paraformaldehyde, followed by permeabilization with 0.4% Triton X-100. Next, the tissue samples and cells were blocked with goat serum (5%). Subsequently, the samples were incubated with macrophage marker CD68 (MCA1957, 1:200 dilution, Bio-Rad, Hercules, CA, USA), endothelial marker CD31 (1:200; Abcam ab9498), and
SRSF10 antibody (1:500, Novus Biologicals, Littleton, CO, NB110-93598) at 4 °C overnight. Then, the samples were probed with secondary antibody (1:500 dilutions) and examined under a confocal laser scanning microscope (FV300, Olympus, Japan).
2.7 RNA sequencing
Total RNA from pretreated HAECs was extracted using the TRIzol reagent and purified with the RNAClean XP Kit (Cat A63987, Beckman Coulter, Inc., Kraemer Boulevard Brea, CA, USA) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). The quality of total RNA samples was determined using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Next, the rRNA was removed from the samples by using the Ribo-Zero rRNA Removal Kit (Epicenter, Madison, WI, USA). Then, the RNA samples were fragmented and reverse-transcribed to cDNA with random primers. After purification, the cDNA libraries underwent a quality check and were sequenced using HiSeq2000 (Illumina, San Diego, CA, USA). Raw reads in FASTQ format were screened, and the adaptor sequences and low-quality reads were deleted. Stringent filtering criteria were adopted to reduce the number of sequencing (RNA-seq) errors. After genome mapping, reads with less than two base mismatches and multiple hits ≤ 2 were retained. Differentially expressed genes were identified as P < 0.05 and fold change (FC) ≥ 2.
2.8 Quantitative real-time PCR (qRT-PCR) analysis
qRT-PCR was performed to validate the RNA-seq data. The sequences of primers are listed in Tab.1. Briefly, the TRIzol (Invitrogen, Carlsbad, CA, USA) reagent was used to extract total RNA from HAECs according to the manufacturer’s protocol. A total RNA sample of 0.5 µg was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The SYBR Green PCR Master Mix Kit (Applied Biosystems) was used to quantify mRNAs with GAPDH or U6 as an internal control. The threshold cycle (CT) was obtained, and relative mRNA levels were calculated using the 2−∆∆Ct method.
2.9 Western blot analysis
Total protein was extracted from ECs using the same procedures as described in detail elsewhere [
17]. The membranes were incubated with primary antibodies against LC3 (4108S; Cell Signaling Technology, Danvers, MA, USA), p62 (39749S; Cell Signaling Technology),
SRSF10 (NB110-93598; Novus Biologicals, Littleton, CO, USA), and GAPDH (Proteintech, Chicago, USA) at 4 °C overnight. After washing with PBS-T three times, the membranes were incubated with the fluorescence-conjugated anti-mouse or rabbit IgG secondary antibody (1:10 000) at room temperature for 1 h. Western blot bands were analyzed using the Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA).
2.10 Luciferase reporter assay
The luciferase reporter assay was performed as previously described [
18]. Briefly, luciferase reporters containing wild-type or mutated 3′-UTR of
SRSF10 were constructed using psi-CHECK2 vectors (Promega, Madison, WI, USA). Detailed sequences of the mutated 3′-UTR of
SRSF10 are shown as follow: AAGACC. Next, 293T cells were seeded in a 24-well plate and co-transfected with 0.5 mg of plasmid, miR-193-5p mi, miR-193-5p inh, or NC using the Lipofectamine 2000 reagent. Renilla luciferase was used as an internal control. Twenty-four hours after transfection, the cells were collected, and firefly and Renilla luciferase activities were evaluated using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
2.11 Transmission electron microscopy (TEM) measurement
Conventional TEM examination was performed as described previously [
19]. In brief, cells were fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated in a graded series of ethanol concentrations, and embedded in embed resin. The ultrathin sections were mounted on copper grids and then double-stained with uranyl acetate and lead citrate. The number of autophagic vacuoles was determined for a minimum of 100 cells. The samples were examined and photographed with a FEI Tecnai spirit transmission electron microscope.
2.12 Statistical analysis
Statistical comparisons among multiple groups were performed using one-way analysis of variance. Student’s t-test was performed for comparisons between the two groups. A two-tailed P < 0.05 was considered statistically significant.
3 Results
3.1 lncR-GAS5 aggravated atherosclerosis in ApoE−/− mice
To investigate the role of lncR-GAS5 in atherogenesis, 8-week-old HFD-fed
ApoE−/− mice were intravenously injected with Lv-GAS5 for 12 weeks, with empty vector (Lv null) as a NC (Fig. S1A). Histological analysis of the aortic root sections was performed. H&E and ORO staining revealed larger atherosclerotic lesions and a marked increase in lipid accumulation in the aortic root of Lv-GAS5-treated
ApoE−/− mice with HFD (Fig.1–1C). The stability of atherosclerotic plaques is positively associated with collagen content [
20]. Our results demonstrated that lncR-GAS5 overexpression markedly decreased collagen deposition in lesions (Fig.1 and 1E). Moreover, analysis of en face aortas revealed a remarkable increase in lesion size after infection with Lv-GAS5 (Fig.1 and 1G). Immunofluorescence staining further revealed that lncR-GAS5 overexpression contributed to atherogenesis (Fig.1 and 1I).
To further confirm the role of lncR-GAS5 in atherogenesis, qRT-PCR was performed to detect its expression in aortas. As shown in Fig. S1B, the mRNA expression of the EC marker CD31 was higher in the intima than in the media and adventitia. Conversely, little smMHC, a smooth muscle cell marker, was detected in the intima, indicating that highly pure ECs were obtained from the aortic intima (Fig. S1C). Compared with that in the intima, lncR-GAS5 expression was lower in the media and adventitia, indicating that lncR-GAS5 is more abundant in ECs (Fig. S1D). Moreover, lncR-GAS5 was predominantly increased in the ECs of ApoE−/− mice fed with HFD (Fig. S1E). Furthermore, lncR-GAS5 overexpression substantially reduced MAP1LC3B and increased P62 mRNA expression levels in the aortic intima (Fig. S1F and S1G). Finally, lncR-GAS5 potentiated the development of abnormal hyperlipidemia, but did not alter the bodyweight, blood pressure, and glucose metabolism of mice (Fig. S2). Taken together, these results reveal that lncR-GAS5 overexpression increases the size of atherosclerotic lesions and fibrous caps, two key determinants of plaque vulnerability.
3.2 lncR-GAS5 impairs autophagy in HAECs
The loss of functional autophagy in the endothelium could promote atherogenesis [
21]. Ox-LDL treatment induced a remarkable dose- and time-dependent increase in lncR-GAS5 expression in HAECs (Fig.2 and 2B). The efficacy of lncR-GAS5 overexpression or knockdown was also analyzed. The lncR-GAS5 plasmid (GAS5-p) and siRNA-2 were selected for subsequent experiments (Fig.2). LncR-GAS5 knockdown promoted autophagic flux by increasing the LC3II/LC3I protein ratio and decreasing p62 protein levels (Fig.2). Rapamycin (0, 30, 50, and 100 nmol/L) substantially increased the LC3II/LC3I ratio and reduced the protein level of p62 in a dose-dependent manner (Fig.2). The overexpression of lncR-GAS5 attenuated rapamycin-induced autophagy (Fig.2), whereas lncR-GAS5 knockdown enhanced autophagy in HAECs (Fig.2 and 2H).
3.3 Role of SRSF10 in mediating the effect of lncR-GAS5 on autophagy
To gain insight into the molecular mechanism by which lncR-GAS5 regulates autophagy, a tailored RNA-seq was performed. HAECs were collected after they were transfected with siRNA against lncR-GAS5 for 24 h. Gene ontology analysis indicated that lncR-GAS5 knockdown resulted in the upregulation of 305 genes and downregulation of 138 genes; these differentially expressed genes have been implicated in biological regulation, cellular processes, metabolic processes, and molecular function regulation (Fig. S3A). Volcano plots show the distribution of these differentially expressed genes (Fig. S3B). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the differentially expressed genes were associated with the immune system, lipid metabolism, and signal transduction (Fig. S3C). We speculated that lncR-GAS5 could regulate the activation of miRNAs, thereby influencing the functional role of downstream target genes during the development of atherosclerosis. Thus, a panel of six top downregulated genes was independently validated using qRT-PCR. Intriguingly, RNA-seq and qRT-PCR analyses revealed that knockdown of lncR-GAS5 inhibited the expression of splicing factor SRSF10 in HAECs (Fig.3). Ox-LDL treatment could upregulate SRSF10 expression in HAECs, which was reversed by rapamycin (Fig.3 and 3C). Moreover, lncR-GAS5 knockdown decreased the protein expression of SRSF10 and P62 and increased the protein expression of LC3II/LC3I, which was reversed by the upregulation of SRSF10 (Fig.3 and 3E).
Then, we detected the effect of lncR-GAS5 overexpression on rapamycin-treated HAECs. LncR-GAS5 overexpression increased the protein levels of SRSF10 and P62, and decreased the protein levels of LC3II/LC3I rapamycin-treated HAECs, which was reversed by the knockdown of SRSF10 (Fig.3 and 3G). Consistently, lncR-GAS5 overexpression also remarkably increased the protein expression of SRSF10 in endothelium-containing plaques (Fig.3). SRSF10 knockdown increased the number of autolysosomes and autophagosomes (Fig.3) and enhanced autophagic vacuole accumulation (Fig.3). These findings suggest that lncR-GAS5 impairs autophagy during atherogenesis, at least partially, by upregulating the expression of endogenous SRSF10.
3.4 SRSF10 is involved in lncR-GAS5-mediated autophagy
Broadly conserved miRNAs that could bind with
SRSF10 were predicted by miRcode and TargetScan, which include miR-9, miR-22, miR-21, miR-193-5p, and miR-222. We then measured the effect of lncR-GAS5 knockdown on the expression of these miRNAs. The results showed that lncR-GAS5 knockdown markedly increased the expression of miR-193-5p without affecting the expression of the other miRNAs (Fig.4). The binding site between
SRSF10 mRNA and miR-193-5p is shown in Fig.4. A luciferase assay suggested that miR-193-5p overexpression inhibited luciferase activity elicited by the vector carrying the 3′-UTR of the
SRSF10 sequence in 293T cells (Fig.4 and 4D). Previous studies revealed that decreased miR-193-5p also inhibits autophagy-related protein expression [
22]. Our results showed that co-incubation with miR-193-5p inh reversed the effect of lncR-GAS5 knockdown on autophagy-related proteins (Fig.4 and 4F). Immunofluorescence staining was performed to determine the repressive effect of miR-193-5p mi on the protein levels of
SRSF10. By contrast, the effect of miR-193-5p was effectively abrogated by its antisense inhibitor (Fig.4). Taken together, the aforementioned results indicate that
SRSF10 is a direct target gene of miR-193-5p and that miR-193-5p expression is mediated by lncR-GAS5.
3.5 miR-193-5p disrupts the effect of lncR-GAS5 on autophagy of HAECs
Incubation with rapamycin for 4 h upregulated the expression of miR-193-5p, whereas incubation with ox-LDL (100 µg/mL) for 24 h reversed this increase, indicating that miR-193-5p is involved in regulating autophagy (Fig.5 and 5B). Next, HAECs were transfected with miR-193-5p, and the changes in autophagy were investigated. The transfection efficiency of miR-193-5p was confirmed by qRT-PCR (Fig.5). Subsequently, immunofluorescence staining revealed that miR-193-5p overexpression enhanced the accumulation of autolysosomes and autophagosomes (Fig.5). Moreover, the upregulation of miR-193-5p increased the LC3II/I ratio, decreased the protein level of P62 (Fig.5), and increased the number of autophagic vacuoles (Fig.5). Based on the above observations, it was concluded that miR-193-5p, an SRSF10 inhibitor, protects against the impairment of autophagy in HAECs mediated by lncR-GAS5.
3.6 miR-193-5p alleviates the development of atherosclerosis
We investigated whether the enhancement of autophagy mediated by miR-193-5p is linked to the shrinking of atherosclerotic plaques. To this end, lentivirus coated with miR-193-5p mi was administered into ApoE−/− mice via tail vein injection, and its effects on atherogenesis after HFD feeding for 12 weeks were evaluated. Strikingly, miR-193-5p overexpression clearly attenuated atherosclerotic lesions and decreased lipid accumulation (Fig.6). Moreover, histological analysis of progressive sections of the en face aortas revealed decreases in lesion size (Fig.6). These results obtained by immunofluorescence staining further confirmed that miR-193-5p overexpression reduced the lesion size in the aortic root (Fig.6). In addition, collagen content drastically increased after the overexpression of miR-193-5p (Fig.6). These findings suggest that forced expression of miR-193-5p can mitigate the progression of atherosclerosis and stabilize aortic plaques.
4 Discussion
In this study, the role of lncR-GAS5 in regulating the pathogenesis of atherosclerosis was characterized. The most salient finding of this study is that overexpressed lncR-GAS5 contributes to accelerating atherogenesis, which is characterized by enhanced lipid accumulation, increased plaque size, and decreased collagen content. LncR-GAS5 could affect atherogenesis by regulating miR-193-5p-mediated repression of the expression of its target, SRSF10 (also known as SRp38), impeding autophagy in HAECs. Collectively, our findings support the idea that targeting lncR-GAS5/miR-193-5p/SRSF10 might be a novel approach to stabilize vulnerable atherosclerotic lesions and retard atherogenesis.
Plasma lncR-GAS5 was found to be increased in atherosclerotic plaque [
23]. Exosomal lncR-GAS5 could promote atherogenesis by inducing the apoptosis of macrophages and vascular ECs [
23]. Moreover, lncR-GAS5 knockdown impeded atherosclerosis progression by inhibiting ABCA1 transcription and reducing THP-1 macrophage lipid accumulation [
24]. LncR-GAS5 overexpression not only increased lesion size but also reduced plaque collagen content. Defective autophagy promotes apoptosis and senescence in ECs, thereby accelerating atherogenesis [
21]. Notably, increased endothelial autophagy hampers the formation of atherosclerotic lesion formation [
25]. Recent investigations have revealed the involvement of lncR-GAS5 in regulating vascular EC function [
26]. Our results indicated that lncR-GAS5 knockdown enhanced autophagic flux and increased the number of autophagic vacuoles, autolysosomes, and autophagosomes in ECs.
At the molecular level, lncR-GAS5 knockdown rescued impaired autophagy and restored the number of autolysosomes and autophagic vacuoles, presumably by downregulating the splicing factor SRSF10, which is abundantly expressed in the endothelium.
SRSF10 is an atypical SR protein that functions as a sequence-specific alternative splicing regulator [
27]. Recently, researchers revealed that the loss of the mitophagy-associated gene Pink1 resulted in the additional downregulation of
SRSF10 [
28]. Thus, the effect of endothelium-enriched
SRSF10 on autophagy was examined. Our findings revealed that
SRSF10 knockdown markedly promoted autophagic flux, which was reversed by the overexpression of lncR-GAS5.
To elucidate how lncR-GAS5 controls the activation of SRSF10, the mRNAs that could bind with lncR-GAS5 were predicted. MiR-193-5p was shown to bind with lncR-GAS5 and SRSF10. Our experiment found that lncR-GAS5 not only reduced endogenous miR-193-5p but also reversed the inhibitory effect of miR-193-5p on SRSF10. Notably, miR-193-5p overexpression promoted autophagic flux. Moreover, forced expression of miR-193-5p shrank atherosclerotic plaques, reduced the number of macrophages, and increased collagen content. In summary, the effects of lncR-GAS5 are likely mediated by the miR-193-5p/SRSF10 axis, resulting in the impairment of endothelial autophagy and atherogenesis. Although the potential role of lncR-GAS5 has been uncovered in atherogenesis, one limitation needs to be pointed out. Our experiments only focused on male animals. The role of lncR-GAS5 in female animals still needs further validation.
In conclusion, lncR-GAS5 overexpression arrested endothelial autophagy through the miR-193-5p/SRSF10 signaling pathway (Fig.7). Thus, miR-193-5p/SRSF10 may serve as a novel treatment target for atherosclerosis.
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