Introduction
Cardiovascular disease (CVD) is one of the leading causes of mortality, with an annual mortality rate of >17.3 million worldwide (1). Atherosclerosis (AS) constitutes a large subgroup of CVD and is the biggest cause of heart attack and stroke, accompanied by inflammation, and characterized by lipid accretion and fibrous cap and necrotic core formation (2). The formation of AS lesions involves a diverse range of cells, including endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (3). It is well-known that vascular SMCs (VSMCs) are the major components of the arterial wall. Previous studies have also demonstrated that the abnormal proliferation and migration of VSMCs are important patho-physiological processes in the development of atherosclerosis and that adequate VSMC function is crucial in protecting the vessel wall against atherosclerosis (4,5). The abnormal proliferation, apoptosis, autophagy, and migration of VSMCs are closely associated to AS advancement (6,7). Depending on this, VSMCs were selected in the present study to explore the role of CTBP1-AS2 on cell function. Autophagy also serves an important role in AS (8). Oxidized low-density lipoprotein (ox-LDL), a primary factor in the promotion of AS progression, causes VSMC proliferation, invasion, and apoptosiss (9). Therefore, the regulatory mechanisms involved in AS were explored using ox-LDL-stimulated VSMCs as an AS cell model.
Long non-coding RNAs (lncRNAs) measure 200 nucleotides (nt) long and are a category of non-coding (nc)RNAs that serve as core modulators in manifold complex diseases, including AS (10,11). lncRNAs associated with AS have received extensive attention due to their involvement in the inflammatory response, lipid metabolism, cell proliferation, adhesion, apoptosis and migration (12). For example, lncRNA p21 exhibits an overtly decreased expression in AS plaques of Apolipoprotein E-/- mice and strengthens p53 activity to curb VSMCs and mononuclear macrophage cells of mice to proliferate and boost their apoptosis (13). The downregulation of lncRNA RNCR3 accelerates AS progression by inhibiting the migration and proliferation of ECs and VSMCs, thereby resulting in their apoptosis in vitro (14). lncRNA CTPB1-AS2 is an lncRNA that acts a novel modulator of cardiomyocyte hypertrophy by regulating TLR4 and an oncogene in papillary thyroid cancer (15). However, its role in AS has not been explored.
MicroRNAs (miRNAs) are small ncRNAs measuring ~22 nt that post-transcriptionally modulate genes (16). Numerous miRNAs are pivotal modulators in AS pathological processes, including immune responses, lipid and cholesterol biosynthesis, endotheliocyte vascular and biological functions, cholesterol effluence, and lipoprotein metabolism (17). miR-195-5p is located on chromosome 17p13.1 and belongs to the miR-15a/b/16/195/497 family (18). miR-195-5p is aberrantly expressed and serve a role as tumor suppressor in a number of types of cancer in humans, including prostate (19) and breast cancer (20), and hepatocellular carcinoma (21); however, its functions in AS is rarely known.
In the present study, lncRNA CTPB1-AS2 was downregulated among patients with AS, and miR-195-5p was identified as a potential target gene. The study aimed to investigate the possible effects and molecular characteristics of CTBP1-AS2 in VSMC progression, to determine the potential targets of AS treatment.
Materials and methods
Clinical samples
The present study was approved by The First Affiliated Hospital of Anhui Medical University. All samples were collected between February 2015 and October 2019. The subjects, aged between 50-70 years (mean ± standard deviation, 61.21±1.302 years), including 21 males and 3 females, provided written informed consent. Then, 10 ml blood samples were collected from 24 patients with untreated AS and 24 healthy volunteers (age range, 50-70 years; mean ± standard deviation, 58.88±1.081 years; 20 males and 4 females) into anticoagulant-free centrifuge tubes in the hospital. The healthy volunteers also provided written informed consent. The enrollment criteria for healthy volunteers were as follows: Individuals without AS, inflammatory diseases, malignant tumors, autoimmune diseases, or recent infection (< 1 month). The collected blood samples were preserved at room temperature for 1 h and centrifuged to extract serum at 1,006 x g, 4°C for 5 min. TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for RNA isolation from the serum.
Cell culture
Human aorta VSMCs (HA-VSMCs) were preserved in F-12 K medium acquired from American Type Culture Collection, and the medium contained 10% fetal bovine saline (Invitrogen; Thermo Fisher Scientific, Inc.), 0.05 mg/ml ascorbic acid, 0.01 mg/ml transferrin, 0.01 mg/ml insulin, 10 ng/ml sodium selenite, 10 mM TES and 10 mM HEPES (Sigma-Aldrich; Merck KGaA), and 0.03 mg/ml endothelial cell growth supplement purchased from Cell Applications Inc., in a humid incubator under 5% CO2 and 37°C.
Cell transfection and treatment
Full length CTBP1-AS2 sequences were amplified via PCR and subcloned into pcDNA3.1 vectors (Invitrogen; Thermo Fisher Scientific, Inc.) to generate pcDNA-CTBP1-AS2 overexpression (OE) plasmids. The small interfering RNAs (siRNAs) specific to CTBP1-AS2, miR-195-5p mimic, and miR-195-5p inhibitor and their corresponding negative controls si negative control (NC), mimic NC, and inhibitor NC were designed and synthesized by Shanghai GenePharma Co. Ltd. Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was utilized for cell transfection following the manufacturer's protocol. HA-VSMCs were treated with ox-LDL (0, 25, 50 and 100 μg/ml; Beijing Biosynthesis Biotechnology Co., Ltd.) for 1 day to determine its effects on CTBP1-AS2 and miR-195-5p expression levels. ROC-325 (MedChemExpress), a novel inhibitor of autophagy, was dissolved in double distilled water (ddH2O) and applied to inhibit cell autophagy. Following treatment with 5 μM ROC-325 for 24 h, the levels of autophagy proteins LC3I/II and Beclin1 were examined.
Reverse transcription-quantitative (RT-q)PCR
TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA isolation following the manufacturer's guidelines. Then, 1 μg RNA from each serum or cell sample was reverse transcribed into first-strand cDNA using RT primers (miR-195-5p and U6 small nucleolar RNA) or random primers (CTBP1-AS2 and β-actin) and M-MLV reverse transcriptase from Invitrogen; Thermo Fisher Scientific, Inc. CTBP1-AS2, autophagy related 14 (ATG14), GAPDH, miR-195-5p and U6 expression levels were examined using qPCR primers and SYBR Green Real-Time PCR Master Mix provided by Toyobo Life Sciences. The 2−ΔΔCq method (22) was used to calculate the relative gene expression normalized by GAPDH and U6. The sequences of the primers were listed below: CTBP1-AS2 forward, 5′-GAG CCC TGA TTT ACC GTC CG-3′ and reverse, 5′-AAGGGGAGACGTAGGCTTCT-3′; ATG14 forward, 5′-CATAACAACCCCGCCTACAC-3′ and reverse, 5′-TGCGTTCAGTTTCCTCACTG-3′; miR-195-5p forward, 5′-GGGGTAGCAGCACAGAAAT-3′ and reverse, 5′-TCCAGTGCGTGTCGTGGA-3′; U6 forward, 5′-CTCGCTTCGGCAGCAGCACATATA-3′ and reverse, 5′-AAATATGGAACGCTTCACGA-3′; GAPDH forward, 5′-GAAGAGAGAGACCCTCACGCTG-3′ and reverse, 5′-ACTGTGAGGAGGGGAGATTCAGT-3′.
Western blot analysis
RIPA lysis buffer (Beyotime Institute of Biotechnology) and a Pierce bicinchoninic acid Protein Assay kit (Thermo Fisher Scientific, Inc.) were used to isolate and quantify whole proteins, respectively. Then, proteins (50 μg) were separated using 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with fat-free milk (5%) for 1 h at room temperature and incubated at 4°C overnight with primary antibodies against the following: Proliferating cell nuclear antigen (PCNA; cat. no. ab92552; 1:1,000), GAPDH (cat. no. ab181602; 1:2,500), proliferation marker protein Ki-67 (cat. no. ab92742; 1:2,000), Beclin-1 (cat. no. ab62557; 1:1,000) and microtubule-associated proteins 1A/1B light chain 3B (LC3; cat. no. ab51520; 1:1,000). All primary antibodies were from Abcam. The membranes were then incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibody (cat. nos. ab97040 and ab97080, respectively; 1:3,000) from Abcam. Clarity Max™ Western ECL Substrate provided by Bio-Rad Laboratories, Inc., was used to amplify certain protein signals. Image J (V1.8.0.112; National Institutes of Health) was used to perform the densitometric analysis.
CCK-8 experiment
Subsequent to seeding onto 96-well plates at a density of ~1x104/well overnight, HA-VSMCs were transfected for 0, 24, 48 or 72 h, and then 10 μl Cell Counting Kit-8 solution (MedChemExpress) was added to each well and incubated at 37°C for 3 h. Cell proliferation was evaluated by measuring the absorbance (A450).
Colony formation experiment
Cells in the logarithmic growth phase were digested into a single cell layer using 0.25% trypsin and then suspended in F-12 K medium. Each group was inoculated with 200 cells per dish. Following cultivation for 15 days in complete medium, the transfected HA-VSMCs were fixed using 75% methanol for 15 min at room temperature and stained with 0.1% crystal violet solution (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min. Finally, after washing, images of the cells were captured and cells were counted using light microscopy (magnification, x10), and the colony-forming efficiency was calculated using the following formula: Colony forming efficiency=clone number/inoculated cell number x100%.
Intracellular protein segregation
A Cytoplasmic and Nuclear RNA Purification kit (Norgen Biotek Corp.) was used for separation and purification of RNAs in the nucleus and cytoplasm of HA-VSMCs according to the manufacturer's guidelines. Next, CTBP1-AS2, GAPDH and U6 expression levels in nuclear and cytoplasmatic segments were independently examined via RT-qPCR.
Bioinformatics analysis
DIANA tools-miRGenv3 (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=mirgenv3%2Findex) was applied to predict the target gene of CTBP1-AS2.
Luciferase reporter assay
PCR amplification was implemented for specific segments of CTBP1-AS2 and ATG14 3′ untranslated regions (UTRs) with miR-195-5p binding sites, and these segments were created into psiCHECK-2 vector (Promega Corporation). Therefore, wild-type lncRNA CTBP1-AS2 and ATG14 reporter plasmids were generated. A QuikChange Multi Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) was used to generate mutated lncRNA CTBP1-AS2 and ATG14 reporters with mutational miR-195-5p binding sites. HA-VSMCs were then transfected with miRNAs or plasmids and the created luciferase reporters, independently. After 48 h, a dual-luciferase reporter assay kit (Promega Corporation) was utilized to detect cell luciferase activity according to the protocols of the manufacturer.
Statistical analysis
SPSS 22.0 (IBM Corp.) was used for statistical analysis of all data. Each experiment was repeated a minimum of three times, and data are presented as mean ± standard error of the mean. Pearson's analysis was performed to analyze the correlation between miR-195-5p and lncRNA CTBP1-AS2. Comparisons between groups were performed using Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
CTBP1-AS2 level is decreased and miR-195-5p is increased in AS serum and HA-VSMCs treated with ox-LDL
CTBP1-AS2 and miR-195-5p expression levels in the serum of patients with AS and HA-VSMCs treated with ox-LDL were examined. RT-qPCR experiments revealed that CTBP1-AS2 level decreased significantly (Fig. 1A) and miR-195-5p level increased significantly (Fig. 1B) in the serum of patients with AS compared with that of healthy volunteers. Treatment with ox-LDL dose-dependently decreased CTBP1-AS2 expression levels (Fig. 1C) and increased miR-195-5p expression levels (Fig. 1D) in HA-VSMCs. CTBP1-AS2 and miR-195-5p may be pivotal factors in AS advancement.
OE of CTBP1-AS2 inhibits proliferation in HA-VSMCs treated with ox-LDL and triggers their autophagy
The effects of CTBP1-AS2 on the proliferation and autophagy of HA-VSMCs treated with ox-LDL was detected by the use of OE-CTBP1-AS2. The RT-qPCR results indicated that OE-CTBP1-AS2 markedly increased CTBP1-AS2 expression compared with the negative control (NC) in HA-VSMCs treated with ox-LDL (Fig. 2A). The CCK-8 assay results suggested that CTBP1-AS2 markedly decreased the level of proliferation in HA-VSMCs treated with ox-LDL compared with the NC (Fig. 2B). This result was corroborated by the results from the colony formation experiment (Fig. 2C). PCNA and Ki-67 expression levels in HA-VSMCs treated with ox-LDL were examined, and their expression levels exhibited a marked decrease subsequent to CTBP1-AS2 overexpression (Fig. 2D and E). The overexpression of CTBP1-AS2 markedly promoted autophagy, and resulted in significant increases in the lipid-modified LC3/GAPDH ratio and in the Beclin-1 protein expression level in HA-VSMCs compared with the NC (Fig. 2F). Rescue experiments were conducted to determine the association between VSMC proliferation and autophagy. As demonstrated in Fig. 2G, treating cells with 5 μM ROC-325, an inhibitor of autophagy, in combination with CTBP1-AS2 OE plasmids partly reversed the ox-LDL-mediated increase in cell proliferation. CTBP1-AS2 inhibited the proliferation of HA-VSMCs treated with ox-LDL by increasing the levels of autophagy.
CTBP1-AS2 prevents miR-195-5p expression by direct interaction
Bioinformatics analysis was used to ascertain the potential targets of CTBP1-AS2. Complementary sites between CTBP1-AS2 and miR-195-5p were identified (Fig. 3A). As demonstrated in Fig. S1, other potential target miRNAs were also detected; however, no significant difference between the patients with AS and the controls was identified (Fig. S1A-F). CTBP1-AS2 was primarily located in the cytoplasm of HA-VSMCs, as verified by the intracellular segregation experiment, indicating the potential interaction between CTBP1-AS2 and miR-195-5p due to their shared location (Fig. 3B). The luciferase reporter assay verified that treatment with the miR-195-5p mimic decreased the luciferase activity in wild-type CTBP1-AS2 reporter plasmid by ~48% compared with that in the scramble control (Fig. 3C). Nevertheless, miR-195-5p did not affect the luciferase activity of the mutated CTBP1-AS2 reporter plasmid, which had mutated assumed binding sites between CTBP1-AS2 and miR-195-5p (Fig. 3C). An RT-qPCR assay was implemented to determine the effects of CTBP1-AS2 on miR-195-5p expression. CTBP1-AS2 upregulation decreased miR-195-5p expression, whereas si-CTBP1-AS2 transfection significantly increased miR-195-5p expression (Fig. 3D and E). miR-195-5p expression exhibited an inverse correlation with CTBP1-AS2 levels in the serum of 24 patients with AS (Fig. 3F). These data confirmed that CTBP1-AS2 inhibited miR-195-5p expression by direct reciprocal action.
miR-195-5p mimic counteracts the effects of CTBP1-AS2 on the levels of proliferation and autophagy in HA-VSMCs treated with ox-LDL
The RT-qPCR assay results demonstrated that the miR-195-5p mimic induced miR-195-5p expression and promoted miR-195-5p expression via CTBP1-AS2 in HA-VSMCs treated with ox-LDL (Fig. 4A). Co-treatment with miR-195-5p and ox-LDL promoted HA-VSMCs to proliferate and form colonies (Fig. 4B and C). The CCK-8, colony formation and western blot assays results showed that the effect of CTBP1-AS2 on cell growth was remarkably abrogated by the miR-195-5p mimic, as demonstrated by increased cell proliferation (Fig. 4B), colony formation (Fig. 4C), and PCNA and Ki-67 expression levels (Fig. 4D). miR-195-5p significantly counteracted CTBP1-AS2-mediated autophagy, as shown by the increased level of the autophagy-associated proteins (Fig. 4E). miR-195-5p can modulate the CTBP1-AS2-mediated effects by inhibiting proliferation and promoting autophagy in HA-VSMCs treated with ox-LDL.
ATG14 is a target of miR-195-5p
Multiple studies have revealed that lncRNAs modulate target mRNA expressions via their function as competing endogenous RNAs (ceRNAs) of miRNAs (23-25). Bioinformatics methods were used to find the potential targets of miR-195-5p. The results revealed that potential binding sequences of miR-195-5p were present in the ATG14 3′UTR (Fig. 5A). Concomitantly, the relative expression levels of other potential target genes in AS were also detected, but no significant difference was observed (Fig. S1G-L). Subsequently, the dual luciferase reporter assay showed that miR-195-5p evidently inhibited the luciferase activity of the wild-type ATG14 reporter plasmid, but did not change that of the mutated ATG14 reporter (Fig. 5B). The data from the clinical samples indicated that ATG14 exhibited a high expression level in the serum of healthy volunteers compared with those of patients with AS (Fig. 5C). The RT-qPCR assay data demonstrated that the ox-LDL-mediated decrease in ATG14 was dose-dependent (Fig. 5D). The RT-qPCR results also showed that ATG14 expression was positively correlated with CTBP1-AS2 expression (Fig. 5E) but inversely correlated with miR-195-5p expression (Fig. 5F) in the serum of 24 patients with AS. In addition, CTBP1-AS2 induced ATG14 expression, and si-CTBP1-AS2 decreased ATG14 expression in HA-VSMCs treated with ox-LDL (Fig. 5G and H). miR-195-5p upregulation decreased ATG14, but its inhibitor increased ATG14 (Fig. 5I and J). Western blot analysis demonstrated that decreased CTBP1-AS2 or miR-195-5p markedly inhibited ATG14 expression compared with their respective control (si-NC or mimic-NC). Ectopically expressed CTBP1-AS2 abrogated the miR-195-5p-mediated inhibition of ATG14 expression in HA-VSMCs when the cells were treated with ox-LDL (Fig. 5K). Collectively, these results suggested that CTBP1-AS2 promoted ATG14 expression by serving as a ceRNA for miR-195-5p in HA-VSMCs.
Discussion
Despite substantial advances in research investigating its diagnosis and treatment methods, AS remains a leading threat to human health with high incidence and mortality rates worldwide (26). ncRNAs, including miRNAs and lncRNAs, exert pivotal functions in AS development (27,28). In the present study, bioinformatics analysis was used to identify the possible targets of CTBP1-AS2 and further investigate its molecular mechanism. The results suggested that miR-195-5p may act as a downstream gene of CTBP1-AS2. miR-195-5p, belonging to the miR-195 family, modulates core processes in cells, including cell cycle progression, proliferation, invasion, migration and differentiation (29,30). miR-195 regulates the proliferation and migration of VSMCs, induces cytokine secretion mode, and prevents neointimal generation in the cardiovascular system (31).
Ox-LDL can trigger AS by promoting EC activation and dysfunction, VSMC behavior, foam cell generation and other mechanisms (32). As a core vascular cell type, VSMCs are implicated in arterial wall reconstruction, which are important for maintaining blood flow in vessels (33,34). As such, the present study was designed to determine the role of CTBP1-AS2 in AS regulation by controlling miR-195-5p in HA-VSMCs treated with ox-LDL. A decreased level of CTBP1-AS2 and increased level miR-195-5p in the serum of patients with AS were detected. Furthermore, when VSMCs were treated with ox-LDL, the decrease of CTBP1-AS2 and increase of miR-195-5p were ox-LDL dose-dependent.
Autophagy is a self-degradative process mediated by lysosomes and has a critical effect on maintaining cell homeostasis (35); it is implicated in multiple states of vascular diseases, such as hypertension, atherosclerosis and restenosis (36). Autophagy restrained the formation of ox-LDL-provoked VSMCs (37). In the present study, it was identified that CTBP1-AS2 was downregulated in AS and over-expressed CTBP1-AS2 could promote autophagy in VSMCs. According to a previous study (38), enhanced autophagy can inhibit AS, and inhibition of autophagy may aggravate the occurrence of AS (35). Therefore, the low expression levels of CTBP1-AS2 may promote the development of AS by inhibiting autophagy. In addition, the abnormal proliferation of VSMCs is an important pathophysiological process in the development of atherosclerosis, and the inhibition of VSMCs proliferation may delay disease progression (39). In the present study, it was also identified that the upregulated CTBP1-AS2 impaired the proliferation of VSMCs; therefore, the downregulation of CTBP1-AS2 may accelerate the progression of AS by promoting the proliferation of VSMCs. The results of the present study also demonstrated that excessive autophagy may trigger autophagy-mediated cell death. However, a systematic review reported that active autophagy is able to induce autophagic death of VSMCs, resulting in decreased synthesis of collagen leading to the destabilization of plaque (8). These contradicting results may be due to the fact that the present study explored the progress of AS from a different perspective. The present study focused on the association between proliferation and autophagy of VSMCs, however the aforementioned systematic review may focus on the relationship between autophagy and the synthesis of collagen, which is related to the stability of plaque. However, the role of autophagy in AS is not clear at present (8,40). In addition, it may exert its roles through different ways. The results of the present study provide new insights concerning the association between autophagy and AS. This topic should also be investigated in more detail to improve understanding on the mechanism of autophagy during AS progression. This may help find a balance point where autophagy inhibits the proliferation of VSMCs but does not cause the destabilization of plaques in the development of AS.
The mechanism of CTBP1-AS2 in AS was verified by determining whether its effects of the proliferation and autophagy of HA-VSMCs was mediated through miR-195-5p. According to the results of the present study, the anti-proliferation and pro-autophagy effects modulated by CTBP1-AS2 were markedly inhibited by the decrease in miR-195-5p expression in HA-VSMCs treated with ox-LDL.
miRNAs modulate the translation or stability of target mRNAs. Therefore, the possible target mRNAs of miR-195-5p were predicted, and the role of ATG14 as the potential target of miR-195-5p was verified by the luciferase reporter assay. ATG14 is an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (41). Autophagy occurs in developing atherosclerotic plaques (42). ATG14 mRNA expression was demonstrated to exhibit a positive correlation with that of CTBP1-AS2 but a negative correlation with that of miR-195-5p in the serum of patients with AS. miR-195-5p upregulation decreased the ATG14 protein expression level, and CTBP1-AS2 increased the level slightly. Therefore, CTBP1-AS2 may increase ATG14 expression via its role as a ceRNA for miR-195-5p in HA-VSMCs.
However, there were certain limitations in the present study; for example, only the proliferation and autophagy of VSMCs were detected in the cell function experiments without cell cycle and apoptosis. However, it is important to note that, to the best of our knowledge, there have only been few studies investigating the association between proliferation and autophagy of VSMCs; therefore, the present study aimed to reveal the association between proliferation and autophagy of VSMCs. Finally, as there was no access to a working flow cytometer, the levels of apoptosis could not be examined. When this has been resolved, it will be included in our future studies.
The results of the present study demonstrated the involvement of CTBP1-AS2/miR-195-5p/ATG14 regulatory axis in the proliferation and autophagy of HA-VSMCs and suggest the potential use of CTBP1-AS2 in AS prevention. However, the use as a drug target and mechanisms of CTBP1-AS2 in VSMCs and AS should be studied further in in vivo animal models.