Article ID: CJ-21-0042
Background: Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be involved in the progression of many cancers; however, the role and mechanisms underlying NEAT1 in abdominal aortic aneurysm (AAA) remain unclear.
Methods and Results: The expression of NEAT1, miR-30d-5p and A disintegrin and metalloprotease 10 (ADAM10) was measured by qRT-PCR and western blot. Functional experiments were conducted by using a CCK-8 assay, EDU assay, flow cytometry, western blot, ELISA, and commercial kits. The target relation was confirmed by dual-luciferase reporter assay and the RIP assay. It was then found that NEAT1 was upregulated in peripheral blood of AAA patients ~3.46-fold, smooth muscle cells (SMCs) isolated from AAA tissues ~2.6-fold and in a hydrogen peroxide (H2O2)-induced injury model of human vascular SMC (HVSMCs) ~2.0- and 3.9-fold at 50 µmol/L and 200 µmol/L H2O2 treatment, respectively. NEAT1 deletion attenuated H2O2-induced cell proliferation promotion (40.0% vs. 74.3%), apoptosis inhibition (25.0% vs. 13.5%), and reduction of inflammatory response and oxidative stress in HVSMCs. Mechanistically, NEAT1 targeted miR-30d-5p to prevent the degradation of its target, ADAM10, in HVSMCs. Further rescue experiments suggested miR-30d-5p inhibition mitigated the effects of NEAT1 deletion on H2O2-induced HVSMCs. Moreover, ADAM10 overexpression counteracted the inhibitory functions of miR-30d-5p on H2O2-evoked HVSMC injury.
Conclusions: NEAT1 promoted H2O2-induced HVSMC injury by inducing cell apoptosis, inflammation and oxidative stress through miR-30d-5p/ADAM10 axis, indicating the possible involvement of NEAT1 in the pathogenesis of AAA.
Abdominal aortic aneurysm (AAA) is an enlarged area in the lower part of the major vessel that supplies blood to the body,1 in which a rupture can cause life-threatening bleeding. Ruptured AAA and the related physiological side-effects lead to an overall mortality rate of >80%, making it the tenth leading cause of death among men aged ≥65 years.2 AAA is characterized by local inflammation, loss of arterial wall integrity, significant extracellular matrix (ECM) degradation and human vascular smooth muscle cell (HVSMC) apoptosis.3 Depletion and apoptosis of HVSMCs are considered to be important factors in the progression of AAA because it eliminates a cell population that promotes connective tissue repair.4,5 Therefore, targeting HVSMC apoptosis may be a potential therapeutic strategy for AAA treatment.
AAA formation starts from localized remodeling and vessel dilation attributable to degeneration of elastin and alterations in collagen proteins within the aortic wall.6 The oxidative stress and associated production of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) are critical mechanisms involved in the pathogenesis of AAA through promoting VSMC apoptosis and ECM degradation.7 The capability of HVSMCs to synthesize collagens and elastin is sharply decreased by oxidative damage, causing the degeneration of aortic walls and eventual rupture.8 In addition, ROS have been detected at all stages of aneurysm development and mediates ECM degradation and remodeling.9 Hydrogen peroxide (H2O2), one type of ROS, has been widely applied to mimic H2O2-induced oxidative stress in vitro and has been revealed to cause vascular injury and promotes AAA formation.8,10,11 Therefore, in the present study, H2O2 was used to induce HVSMC injury to mimic ROS-induced pathological characteristics of AAA for further in vitro experiments.
Long non-coding RNAs (lncRNAs) are a common type of transcripts longer than 200 nucleotides. It has been documented that lncRNAs act as critical modulators in many diseases by regulating diverse biological processes.12,13 Recently, increasing evidence has shown that lncRNAs participate in the regulation of HVSMCs apoptosis and proliferation during AAA formation and are critical determinants in AAA.14,15 LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is a well-recognized oncogene, and has been revealed to implicate in several biological processes to promote cell development and progression in most human malignancies.16,17 Recently, NEAT1 was found to be involved in postischemia myocardial remodeling, and promoted intima thickening or even vascular occlusion by controlling the phenotype conversion of VSMCs;18,19 however, the roles of NEAT1 in AAA remain elusive.
It has been reported lncRNA serves as a “microRNA (miRNA) sponge” to modulate miRNA expression in various diseases, including AAA.15 To date, a growing number of studies have identified miRNAs to be involved in the progression and formation of AAA.20,21 For example, miR-24 was demonstrated to limit aortic vascular inflammation and murine AAA development.22 Shi et al revealed that miR-144-5p limited the formation of AAA by reducing Ang II-induced aortic dilatation and elastic degradation, and mitigating M1 macrophage-associated inflammation.23 MiR-30d-5p is a functional miRNA and plays important roles in the carcinogenesis of several cancers.24 In addition, a recent study discovered that miR-30d-5p was reduced in the peripheral blood of patients with AAA.25 Thus, miR-30d-5p may be involved in the formation of AAA. The A disintegrin and metalloprotease 10 (ADAM10) is a member of the ADAM family, which are cell surface proteins with a unique structure possessing both potential adhesion and protease domains, and has been found to closely relate to the development of AAA,26,27 but its regulatory factor is still indistinct.
In the present study, H2O2 was used to evoke HVSMC injury to mimic ROS-induced pathological characteristics of AAA in vitro; the potential biological function of NEAT1 in AAA development was then explored. In addition, a rescue experiment was conducted to evaluate the regulatory effects of NEAT1, miR-30d-5p and ADAM10 on the progression of AAA.
Peripheral blood samples and human AAA tissue samples were collected from 20 AAA patients aged from 43 to 70 years (9 males and 11 females) who received infrarenal aorta replacement procedures at the Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology. When technically feasible and without jeopardizing the technical success of the procedure and the expected patient outcomes, non-aneurysmatic aortic samples of the aneurysm neck were also collected for use as controls (n=20). The control peripheral blood samples were obtained from 20 healthy donors, including 9 males and 11 females, and the age range was 40–62 years. These healthy donors were to match the age and gender distribution of the patient group, and exclusion criteria for the control group included cancer, drug history, infection, or any other immune-related disease that might have influenced the study. The demographic and clinical characteristics of all participants are shown in Supplementary Table. Written informed consent was obtained from all participants, and the study protocols were approved by the ethics committee of the Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology (IRB number: 20190316). Animal studies were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and the Basel Declaration. All animals received humane care according to the National Institutes of Health (USA) guidelines.
Cell Culture and Treatment of H2O2Human AAA tissue samples (5 total) and non-AAA aortic tissues (5 total) were cut into 1- to 2-mm3 pieces and placed in a sterile tube with ice-cold phosphate-buffered saline (PBS), and then were used to isolate arterial SMC using enzymatic hydrolysis, as previously described.28 To perform in vitro analysis, HVSMCs were obtained from the Shanghai Academy of Life Science (Shanghai, China) and grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBA, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin at 37℃ with 5% CO2. The HVSMCs were treated with H2O2 at different concentrations (50 µmol/L and 200 µmol/L) (30% w/w solution; Sigma, St. Louis, MO, USA) and cells in the control group were treated with the same medium without H2O2.
Cell TransfectionThe small interfering RNA (siRNA) sequences targeting NEAT1 (si-NEAT1), siRNA negative control (Scramble), pcDNA (Lnc-NC or vector), pcDNA-NEAT1 overexpression vector (NEAT1), and pcDNA-ADAM10 overexpression vector (ADAM10) were purchased from Genepharma (Shanghai, China). The miR-30d-5p mimics (miR-30d-5p), miR-30d-5p inhibitor (anti-miR-30d-5p), and the corresponding control vectors (miR-NC or anti-NC) were obtained from RIBOBIO (Guangzhou, China). The transfection of plasmids or miRNAs was carried out using LipofectamineTM 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA).
Quantitative Real-Time Polymerase Chain ReactionTrizol reagent (Invitrogen) was used for total RNA extraction from cells and serum according to the manufacturer’s instructions. The cDNA was synthesized using the cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using SYBR Select Master Mix (Applied Biosystems) and special primers on an ABI 7900-fast thermocycler. GAPDH or U6 was used as an internal control, and relative expression was evaluated using the 2−∆∆Ct method. The same experiment was repeated 3 times, and the average was taken.
Cell Viability AnalysisThe cell viability was measured by a cell counting Kit-8 assay (CCK-8; Beyotime, Shanghai, China). Briefly, transfected or untransfected HVSMCs were seeded in 96-well plates at a density of 5×103 per well and then treated with H2O2. After that, 10 µL CCK-8 solution was added to each well and incubated at 37℃ with 5% CO2 for 90 min, and the absorbance at 450 nm was measured using a microplate reader. All experiments were repeated 3-fold independently.
EdU Incorporation AssayTransfected or untransfected HVSMCs were seeded into 96-well plates at a density of 1×104 cells/well, followed by treatment with H2O2. Then, cell proliferation was evaluated following the manufacturer’s instructions for the EdU incorporation assay kit (RiboBio, Guangzhou China) and images were obtained using a fluorescence microscope.
Flow CytometerTransfected or untransfected HVSMCs were treated with H2O2 for 6 h, then the apoptosis of HVSMCs was examined by an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. Subsequently, apoptotic analysis was carried out using a BD FACSCanto II flow cytometer (BD Biosciences).The experiment was repeated 3-fold.
Assessment of ROS GenerationThe generation of intracellular ROS was assessed with the help of 2’, 7’-DCF diacetate (DCFH-DA; Sigma). HVSMCs, following assigned transfected or not, were treated with H2O2 for 6 h. Then, cells were incubated in fresh EBM-2 supplement 20 µmol/L DCFH-DA for 30 min at 37℃. After discarding the DCFH-DA-containing medium, ROS formation was stimulated by H2O2 (final concentration: 1 mmol/L) at 37℃ for 6 h. Before and 6 h after H2O2 addition, a fluorescence microplate reader was used to measure the fluorescence levels of the samples at 488 nm excitation and 525 nm emission wavelengths. Triplicate individual experiments were performed in this study.
Measurement of MDA and SODCommercial MDA and SOD determination kits were obtained from Sangon Biotech (Shanghai, China). Transfected or untransfected HVSMCs were treated with H2O2 for 6 h, then HVSMCs in complete medium were centrifuged at 4,000 g for 10 min at 37℃. The supernatant of HVSMCs were collected and levels of MDA and SOD were detected according to the manufacturer’s protocols for commercial assay kits. All values were normalized to the total protein levels and the experiment was repeated 3-fold.
Enzyme-Linked Immunosorbent Assay (ELISA)Transfected or untransfected HVSMCs were treated with H2O2 for 6 h, then the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 from the culture supernatants of HVSMCs were detected using the commercial ELISA kits (R&D Systems, Minneapolis, Minnesota, USA).
Western Blot AssayTotal proteins were extracted from cells using RIPA Lysis Buffer (Beyotime) according to the manufacturer’s protocol and quantified with a BCA Protein Assay Kit (Beyotime). The protein samples were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (PVDF; Merck Millipore, Billerica, MA, USA) and then blocked with 5% skimmed milk at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibodies against cleaved caspase-3, total-caspase-3, Bcl-2, ADAM10 and GAPDH at 4℃ overnight, followed by incubation with the secondary HRP-conjugated antibody for 2 h at room temperature. Finally, the blots were visualized by using ECL Western Blotting Substrate (Beyotime). The results represent as the average of 3 independent replicates.
Dual-Luciferase AssayThe NEAT1/ADAM10 3’-UTR containing wild-type (wt) or mutant (mut) binding sequences of miR-30d-5p were amplified and cloned into the pmirGLO basic vectors (Promega, Shanghai, China). Then, HVSMCs were co-transfected with constructed vectors and miR-30d-5p mimics, anti-miR-30d-5p, miR-NC, or anti-NC using Lipofectamine 2000 reagent. After 48 h transfection, the luciferase activity was assessed by a Dual Luciferase assay Kit (Promega). Each group was run in triplicate in 24-well plates.
RNA Immunoprecipitation (RIP) AssayAn RIP assay was carried out by using a Magna RNA immunoprecipitation kit (Millipore). HVSMCs were lysed in RIP buffer, then the lysis was incubated with magnetic beads coated with anti-Ago2 or IgG antibody. Finally, the enrichment was analyzed by qRT-PCR. Experiments were performed 3-fold.
Statistical AnalysisStatistical analysis was conducted using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA). The data were expressed as mean±standard deviation (SD). Differences were analyzed with a Student’s t-test between 2 groups or 1-way analysis of variance (ANOVA) among multiple groups. The correlation analysis among NEAT1, ADAM10 and miR-30d-5p was analyzed by using Spearman’s rank correlation. P<0.05 was considered a statistically significant difference.
In order to explore the potential effects of NEAT1 in AAA, the expression of NEAT1 was firstly measured using qRT-PCR. A significant increase of the NEAT1 level in peripheral blood of patients with AAA was detected compared with control peripheral blood samples (Figure 1A). Then, the sample tissues of arterial aneurysm and adjacent normal tissues were collected (Figure 1B). Similarly, NEAT1 expression was also elevated in SMCs isolated from human AAA walls (Figure 1C), and after decreasing the levels of NEAT1 in isolated cells by transfecting them with si-NEAT1 (Figure 1C), we found NEAT1 knockdown promoted cell viability (Figure 1D). Thus, NEAT1 may be involved in the end stage of AAA development. Then, H2O2 was used to evoke HVSMC injury to mimic ROS-induced pathological characteristics of AAA in vitro; also, NEAT1 expression was elevated in H2O2-induced HVSMCs, especially in HVSMCs treated with 200 µmol/L H2O2, relative to the control (Figure 1E).
Expression of nuclear paraspeckle assembly transcript 1 (NEAT1) in peripheral blood of abdominal aortic aneurysm (AAA) patients and hydrogen peroxide (H2O2)-treated human vascular smooth muscle cells (HVSMCs). (A) The level of NEAT1 was measured in peripheral blood of AAA and non-AAA patients by quantitative real-time polymerase chain reaction (qRT-PCR). (B) The representative aortic histopathology from each group. (C) The level of NEAT1 was measured in VSMCs isolated from human AAA walls using qRT-PCR after transfection with Scramble or si-NEAT1. (D) The viability of isolated VSMCs was detected by using a CCK-8 assay after transfection. (E) The level of NEAT1 was measured in HVSMCs treated without or with H2O2. *P<0.05.
Subsequently, the H2O2-induced HVSMCs injury model was established by simulating pathological conditions of AAA in vitro. After treatment with different concentrations of H2O2 for 6 h, the proliferation of HVSMCs was significantly decreased, evidenced by using a CCK-8 assay and EDU assay (Figure 2A,B).The results of flow cytometry showed that H2O2 treatment obviously increased the total apoptosis rate in HVSMCs (Figure 2C). ELISA analysis suggested the levels of TNF-α, IL-6, and IL-1β in HVSMCs was remarkably elevated by H2O2 treatment (Figure 2D–F). It is well known that ROS, MDA, and SOD are the markers of oxidative damage. Here, we determined their contents in H2O2-treated HVSMCs. Results showed treatment of H2O2 enhanced ROS production and MDA level, but reduced SOD content in HVSMCs (Figure 2G–I). In addition, results from western blot analysis indicated the level of cleaved-caspase-3 was enhanced, but Bcl-2 was suppressed by H2O2-treatment; however, the level of total caspase-3 was not changed (Figure 2J), further indicating H2O2 induced apoptosis of HVSMCs.
Hydrogen peroxide (H2O2) treatment inhibits cell proliferation as well as induces cell apoptosis, inflammation, and reactive oxygen species (ROS) production in human vascular smooth muscle cells (HVSMCs). HVSMCs were incubated with 50 µmol/L and 200 µmol/L H2O2 for 6 h. (A,B) Cell proliferation was measured by a CCK-8 assay and EDU assay. (C) Cell apoptosis was tested via flow cytometry. (D–F) Enzyme-linked immunosorbent assay (ELISA) analysis of TNF-α, IL-6, and IL-1β levels. (G) The generation of reactive oxygen species (ROS) was assessed using 2’,7’-DCF diacetate. (H,I) Contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected using commercial kits. (J) Apoptosis-related protein expression was measured by western blot. *P<0.05.
To elucidate the potential biological function of NEAT1 in AAA, HVSMCs were transfected with Scramble or si-NEAT1 for 24 h before 200 µmol/L H2O2 treatment, and then a decrease of NEAT1 expression in the si-NEAT1 group was verified compared with that in the Scramble group (Figure 3A). Subsequently, we discovered NEAT1 silencing alleviated H2O2 treatment-induced inhibition of cell proliferation (Figure 3B,C), enhancement of cell apoptosis (Figure 3D,K), elevation of TNF-α, IL-6, and IL-1β levels (Figure 3E–G), production of ROS and MDA (Figure 3H,J), as well as reduction of SOD (Figure 3K) in HVSMCs.
Nuclear paraspeckle assembly transcript 1 (NEAT1) silencing attenuates hydrogen peroxide (H2O2)-induced human vascular smooth muscle cell (HVSMC) injury. HVSMCs were transfected with si-NEAT1 or Scramble, and then treated with 200 µmol/L H2O2 for 6 h. (A) The expression of NEAT1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) to detect transfection efficiency. (B,C) Cell proliferation was measured by a CCK-8 assay and EDU assay. (D) Cell apoptosis was tested via flow cytometry. (E–G) Enzyme-linked immunosorbent assay (ELISA) analysis of TNF-α, IL-6, and IL-1β levels. (H) The generation of reactive oxygen species (ROS) was assessed using 2’,7’-DCF diacetate. (I,J) Contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected using commercial kits. (K) Apoptosis-related protein expression was measured by western blot. *P<0.05.
To further investigate the underlying molecular mechanisms by which si-NEAT1 mediated protective effects on the HVSMC injury model, the potential targets were predicted with the help of the Encori database, and miR-30d-5p was found to have putative binding sites in NEAT1 (Figure 4A). Then the dual-luciferase reporter assay was performed and we found miR-30d-5p mimics reduced the luciferase activities of the NEAT1-wt reporter vector but not the mutant reporter vector in HVSMCs (Figure 4B). Furthermore, the interaction between miR-30d-5p and NEAT1 was also confirmed by using a RIP assay due to the enrichment of miR-30d-5p and NEAT1 expression after Ago2 RIP in HVSMCs (Figure 4C). In addition, we observed the expression of miR-30d-5p was inhibited by NEAT1 overexpression, but was promoted by a NEAT1 decrease in HVSMCs (Figure 4D). Subsequently, the expression of miR-30d-5p was detected and we found miR-30d-5p was downregulated in peripheral blood of patients with AAA and H2O2-induced HVSMCs (Figure 4E,F), which was negatively correlated with NEAT1 expression in AAA patients (Figure 4G).
Nuclear paraspeckle assembly transcript 1 (NEAT1) directly binds to miR-30d-5p and inhibits miR-30d-5p expression. (A) The putative binding sites of NEAT1 and miR-30d-5p are shown. (B,C) The interaction between NEAT1 and miR-30d-5p was confirmed by using a dual-luciferase reporter assay and a RNA immunoprecipitation (RIP) assay. (D) The expression of miR-30d-5p was measured in human vascular smooth muscle cells (HVSMCs) using quantitative real-time polymerase chain reaction (qRT-PCR) after transfection with lnc-NC, NEAT1, si-NEAT1 or Scramble. (E,F) The levels of miR-30d-5p were measured in peripheral blood of AAA patients and hydrogen peroxide (H2O2)-treated HVSMCs by qRT-PCR. (G) The correlation between NEAT1 and miR-30d-5p was analyzed using Spearman’s rank correlation. *P<0.05.
Based on the relationship between NEAT1 and miR-30d-5p, we further investigated whether miR-30d-5p was involved in the action of NEAT1 on H2O2-treated HVSMCs. HVSMCs were transfected with si-NEAT1 + anti-NC, or si-NEAT1 + anti-miR-30d-5p for 24 h prior to 200 µmol/L H2O2 treatment. Immediately, qRT-PCR analysis showed that NEAT1 deletion promoted miR-30d-5p expression in H2O2-treated HVSMCs, whereas this promotion was reversed by the inhibition of miR-30d-5p, indicating the successful transfection (Figure 5A). After treatment with 200 µmol/L H2O2, we found co-transfection of NEAT1 siRNA and the miR-30d-5p inhibitor led to a decreased proliferation rate (Figure 5B,C) and increased apoptosis rate in HVSMCs (Figure 5D,K). In addition, miR-30d-5p inhibition attenuated NEAT1 knockdown-evoked reduction of TNF-α, IL-6, IL-1β (Figure 5E–G), ROS (Figure 5H) and MDA levels (Figure 5J), as well as elevation of the SOD level (Figure 5I) in HVSMCs.
MiR-30d-5p inhibition reverses the protective effects of nuclear paraspeckle assembly transcript 1 (NEAT1) deletion on hydrogen peroxide (H2O2)-induced human vascular smooth muscle cells (HVSMC) injury. HVSMCs were transfected with si-NEAT1 + anti-NC, or si-NEAT1 + anti-miR-30d-5p for 24 h, followed by 200 µmol/L H2O2 treatment. (A) Transfection efficiencies were confirmed using quantitative real-time polymerase chain reaction (qRT-PCR). (B,C) Cell proliferation was measured by a CCK-8 assay and an EDU assay. (D) Cell apoptosis was tested via flow cytometry. (E–G) Enzyme-linked immunosorbent assay (ELISA) analysis of TNF-α, IL-6, and IL-1β levels. (H) The generation of reactive oxygen species (ROS) was assessed using 2’,7’-DCF diacetate. (I,J) Contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected using commercial kits. (K) Apoptosis-related protein expression was measured by western blot. *P<0.05.
Immediately, the target genes of miR-30d-5p were searched using the Encori database, and we found ADAM10 contained the putative binding sites of miR-30d-5p (Figure 6A). Then dual-luciferase reporter assay analysis exhibited miR-30d-5p mimic reduced the luciferase activities of the ADAM10-wt reporter vector but not mutant reporter vector, whereas anti-miR-30d-5p transfection showed the opposite effects on luciferase activities in HVSMCs (Figure 6B), indicating the interaction between miR-30d-5p and ADAM10. In addition, the RIP assay showed a significant enrichment of ADAM10 expression after Ago2 RIP, whereas its efficacy was lost in response to IgG RIP (Figure 6C). Moreover, we also verified ADAM10 was inhibited by miR-30d-5p mimic, but enhanced by anti-miR-30d-5p (Figure 6D).
A disintegrin and metalloprotease 10 (ADAM10) is a target of miR-30d-5p, and nuclear paraspeckle assembly transcript 1 (NEAT1) indirectly regulates ADAM10 expression by sponging miR-30d-5p. (A) The putative binding sites of ADAM10 3’-UTR and miR-30d-5p are presented. (B,C) The interaction between ADAM10 and miR-30d-5p was verified using a dual-luciferase reporter assay and a RNA immunoprecipitation (RIP) assay. (D) The protein of ADAM10 was detected in human vascular smooth muscle cells (HVSMCs) transfected with NC, miR-30d-5p, anti-NC, and anti-miR-30d-5p using western blot. (E,F) The mRNA and protein levels of ADAM10 were measured in peripheral blood of abdominal aortic aneurysm (AAA) patients and healthy persons using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. (G) The protein level of ADAM10 in hydrogen peroxide (H2O2)-treated HVSMCs and controls was examined using western blot. (H,I) The correlation between ADAM10 and miR-30d-5p or NEAT1 was analyzed by Spearman’s rank correlation. (J) The expression of ADAM10 was determined using western blot in HVSMCs transfected with Scramble, si-NEAT1, si-NEAT1 + anti-NC, or si-NEAT1 + anti-miR-30d-5p. *P<0.05.
Subsequently, the expression of ADAM10 was detected and we observed that ADAM10 was upregulated in peripheral blood of patients with AAA both at mRNA and protein levels (Figure 6E,F). Also, its expression was increased in H2O2-induced HVSMCs (Figure 6G). Moreover, a negative correlation between ADAM10 and miR-30d-5p expression (Figure 6H), and a positive correlation between ADAM10 and NEAT1 expression (Figure 6I) in AAA patients were discovered. Furthermore, co-expression analysis indicated NEAT1 silencing reduced ADAM10 expression, whereas this reduction could be rescued by following miR-30d-5p inhibition (Figure 6J).
MiR-30d-5p Protects HVSMCs From H2O2-Induced Injury Through Targeting ADAM10To explore whether miR-30d-5p mediated H2O2-induced HVSMC injury by modulating ADAM10, HVSMCs were transfected with miR-30d-5p + vector, or miR-30d-5p + ADAM10 prior to H2O2 treatment and we found the protein level of ADAM10 was inhibited by overexpressed miR-30d-5p, but was promoted by ADAM10 upregulation, suggesting successful transfection (Figure 7A). After that, a rescue assay implied that overexpressed miR-30d-5p attenuated H2O2-induced HVSMC proliferation inhibition (Figure 7B,C) and apoptosis promotion (Figure 7D,K), which were reversed by ADAM10 overexpression (Figure 7B–D,K). Furthermore, miR-30d-5p overexpression caused the reduction of TNF-α, IL-6, IL-1β release, whereas co-transfection of ADAM10 led to an opposite effect (Figure 7E–G). Additionally, ADAM10 overexpression abated the miR-30d-5p overexpression-mediated decrease of ROS and MDA levels, as well as the increase of the SOD level in HVSMCs.
MiR-30d-5p protects human vascular smooth muscle cells (HVSMCs) from hydrogen peroxide (H2O2)-induced injury through targeting A disintegrin and metalloprotease 10 (ADAM10). HVSMCs were transfected with miR-30d-5p + vector, or miR-30d-5p + ADAM10 and then treated with 200 µmol/L H2O2 for 6 h. (A) The expression of ADAM10 protein was detected by western blot for the verification of transfection efficiency. (B,C) Cell proliferation was measured by a CCK-8 assay and an EDU assay. (D) Cell apoptosis was tested via flow cytometry. (E–G) Enzyme-linked immunosorbent assay (ELISA) analysis of TNF-α, IL-6, and IL-1β levels. (H) The generation of reactive oxygen species (ROS) was assessed using 2’,7’-DCF diacetate. (I,J) Contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected using commercial kits. (K) Apoptosis-related protein expression was measured by western blot. *P<0.05.
The pathological processes of upregulation of proteolytic pathways, oxidative stress, loss of the arterial wall matrix, apoptosis and inflammation are the main causes implicated in the formation of AAA.29 Recently, a growing body of evidence reported that an aberrantly expressed lncRNA pattern was involved in the pathogenesis of various cardiovascular conditions, including AAA.15,30 For instance, lncRNA SENCR inhibited AAA formation by reducing apoptosis of SMCs and ECM degradation.31 PVT1 contributed to VSMC apoptosis, ECM disruption and vascular inflammation in a murine AAA model.32 LncRNA GAS5 promoted SMC apoptosis and suppressed its proliferation by acting as a sponge of miR-21 to induce the formation of AAA.15 This previous evidence indicates lncRNAs are critical regulators of AAA formation and may be effective therapeutic targets for AAA treatment. In this study, we demonstrated NEAT1 played an important role in HVSMC survival. NEAT1 was highly expressed in both peripheral blood of AAA patients and in a H2O2-induced AAA cell model. NEAT1 deletion reversed H2O2-induced HVSMC apoptosis, oxidative stress, and inflammation. In addition, NEAT1 was also elevated in SMCs isolated from human AAA walls, and NEAT1 knockdown promoted cell viability. Thus, abnormal expression of NEAT1 might be associated with AAA.
MiR-30d-5p, a member of the miRNA family, has been revealed to act as a tumor suppressor to inhibit tumor cell proliferation, growth and metastasis through regulating the target mRNA in various cancers, such as gallbladder carcinoma,33 non-small cell lung cancer,34 prostate cancer35 and colon cancer.36 However, deregulation of miR-30d-5p was also reported to be associated with human cardiovascular disease; for example, miR-30d-5p was shown to serve as a diagnostic predictor of AMI and might have a higher diagnostic value than cTnI.37 MiR-30d-5p had a moderate diagnostic value for diffuse myocardial fibrosis and might be a biomarker for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy.38 Additionally, Marques et al proved miR-30d-5p might contribute to an active process in disease pathogenesis in the failing heart.39 In the present study, miR-30d-5p was decreased in peripheral blood of patients with AAA and in a H2O2-induced injury model of HVSMCs. Additionally, functional experiments showed miR-30d-5p protected HVSMCs from H2O2-induced cell apoptosis, oxidative stress, and inflammation. Subsequently, according to the prediction of the Encori database, miR-30d-5p was a target of NEAT1, and a rescue assay suggested miR-30d-5p inhibition reversed NEAT1 deletion-mediated proliferation acceleration, as well as the inhibition of apoptosis and ROS production in H2O2-induced HVSMCs. Thus, a NEAT1/miR-30d-5p network was identified in H2O2-induced HVSMC injury models.
ADAM10 is an important membrane-bound sheddase of the ADAM family and is essential for development because of the requirements for the proper functioning of the HER2 receptor, Notch, classic cadherins or Eph/ephrin.40 Recently, Geng et al indicated an elevated expression of ADAM10 in an established thoracic aortic aneurysm (TAA) model after periarterial CaCl2 exposure in rats, and highly expressed ADAM-10 played a potential role in TAA formation.41 Additionally, Jiao et al revealed that the ADAM10 level was significantly increased in angiotensin II-induced murine AAA specimens, and miR-103a could inhibit AAA growth via targeting ADAM10.27 In this study, ADAM10 was confirmed to be a target of miR-30d-5p by using bioinformatics analysis, and ADAM10 overexpression counteracted the protective effects of miR-30d-5p on H2O2-induced HVSMC dysfunction. Therefore, the miR-30d-5p/ADAM10 axis in HVSMCs was identified. In addition, we also observed that miR-30d-5p was negatively regulated by NEAT1, whereas ADAM10 was positively regulated by it, and NEAT1 served as a sponge of miR-30d-5p to regulate ADAM10 expression in HVSMCs.
In conclusion, this study demonstrated that NEAT1 aggravated apoptosis and inflammation in HVSMCs upon oxidative stress by the miR-30d-5p/ADAM10 axis (Figure 8). These data suggested the potential involvement of NEAT1 in AAA formation and progression, suggesting that a NEAT1 antagonist can act as potential biomarkers and make promising candidates for therapeutic intervention in AAA.
Schematic model showing the role of nuclear paraspeckle assembly transcript 1 (NEAT1) in hydrogen peroxide (H2O2)-induced human vascular smooth muscle cells (HVSMC) injury. NEAT1 promoted H2O2-induced HVSMC injury by regulating the miR-30d-5p/A disintegrin and metalloprotease 10 (ADAM10) axis through the induction of cell apoptosis, inflammation and oxidative stress.
None.
This study received no funding.
The authors declare that they have no competing interests.
The present study was approved by the ethical review committee of the Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology (Reference number: 20190316). Written informed consent was obtained from all enrolled patients.
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.
Conceptualization and Methodology: Z. Zheng, Z. Zha and T.X.; Formal analysis: Z. Zha, T.X. and Y.P.; Data curation, Validation and Investigation: F.Z., Z. Zheng and T.X.; Writing – original draft preparation and Writing - review and editing: F.Z., Z. Zheng, Z. Zha and Y.P.; Approval of final manuscript: all authors.
None.
Please find supplementary file(s);
http://dx.doi.org/10.1253/circj.CJ-21-0042
