Long Noncoding RNA SBF2-AS1 Promotes Abdominal Aortic Aneurysm Formation through the miRNA-520f-3p/SMARCD1 Axis.

Abdominal aortic aneurysm (AAA) is a chronic vascular inflammatory disease. The regulatory mechanisms during AAA formation remain unclear. Bone marrow stem cells (BMSCs) are pluripotent cells capable of regulating the progression of various diseases by delivering exosomes and exosomal lncRNAs. In this study, we investigated its function in AAA by isolating BMSC exosome-derived lncRNA SBF2-AS1. The results showed that BF2-AS1 could be transferred to vascular smooth muscle cells (VSMCs) and human aortic VSMCs (HASMCs) via BMSC-derived exosomes. Depletion of SBF2-AS1 enhanced the cell viability and proliferation of VSMCs. Conversely, SBF2-AS1 knockdown inhibited VSMC apoptosis. Caspase-3 activity was inhibited by depletion of SBF2-AS1, whereas overexpression of SBF2-AS1 in VSMC promoted Caspase-3 activity. SBF2-AS1 enhances SMARCD1 expression by forming miR-520f-3p in VSMC and HASMC. Overexpression of SMARCD1 or miR-520f-3p inhibitor reversed cell viability and caspase-3 activity mediated by SBF2-AS1 depletion in VSMC and HASMC. Therefore, BMSC exosome-derived SBF2-AS1 promotes AAA formation through the miRNA-520f-3p/SMARCD1 axis. Targeting SBF2-AS1 could serve as a promising therapeutic strategy for AAA.

1. Introduction

Abdominal aortic aneurysm (AAA) is a localized dilatation or bulge of the abdominal aorta and is an important cause of sudden death in the elderly population [1-4]. AAA could be a life-threatening disease when the abdominal aorta ruptures [4]. Studies have indicated that apoptosis of vascular smooth muscle cells (VSMCs) contributes to AAA progression owing to the impaired connective tissue repair [5, 6]. Despite the improved survival following the advanced diagnostic and therapeutic approaches, the mortality of AAA still climbs [7]. Therefore, it is urgent to explore the elaborate pathogenesis of AAA [8].

Bone marrow stem cells (BMSCs) are a group of pluripotent cells capable of differentiating into a variety of cells, including osteoblasts and adipocytes [9, 10]. Accumulating evidence has demonstrated the regulatory effects of BMSCs in various diseases, especially cancers and cardiovascular diseases [10, 11]. It is recently suggested that BMSCs mainly communicate with surrounding cells and function via secreting extracellular vesicles (EVs) [12]. Exosomes are a widely studied form of EV with diameters ranging from 40 to 100 nm. Exosomes are capable of delivering a variety of signaling molecules, including RNA, DNA, and proteins [13]. Among which, long noncoding RNAs (lncRNAs) are RNAs with a length over 200 nucleotides that do not translate to proteins [14]. lncRNA SBF2-AS1 is recently reported as an activator in several cancers including lung cancer, pancreatic cancer, and cervical cancer [15-18]. Moreover, lncSBF2-AS1 was also reported to be transferred by exosomes to enhance drug resistance in glioblastoma [19]. However, the role of lncSBF2-AS1 in AAA has not been elucidated.

The most common function of lncRNAs is to act as a sponge for microRNAs (miRNAs), which subsequently interact with the 3′UTR regions of targeted mRNAs and repress gene expression [20]. For example, SBF2-AS1 sponges miR-143 to release RRS1 in breast cancer cells and promotes cell proliferation [21]. miR-520f-3p is a reported tumor suppressor and potential prognostic indicator in several cancers such as cholangiocarcinoma, glioblastoma, gastric cancer, and hepatocellular carcinoma [22-24]. In this study, we aimed to determine the role of BMSC-derived exosomes in AAA progression. Our results suggested that exosomal SBF2-AS1 promotes VSMC proliferation by sponging miR-520f-3p and increasing the expression of the chromatin remodeling protein SMARCD1. Our work may provide novel evidence to support exosomal lncRNAs as a therapeutic manner for AAA.

2. Materials and Methods

2.1. Cell Culture

Mouse vascular smooth muscle cells (VSMCs) and human aortic VSMCs (HASMCs) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Sigma, USA). Cells were cultured in a 37°C incubator filled with 5% CO2.

2.2. Cell Transfection

The small hairpin RNAs (shRNAs) targeting SBF2-AS1 (shSBF2-AS1-1 and shSBF2-AS1-2), miR-520f-3p mimics, and inhibitors and the corresponding negative controls (NCs) were synthesized by RiboBio (China). The overexpressing plasmids targeting SBF2-AS1 or SMARCD1 and the empty vector were obtained from RiboBio (China). Cell transfection was performed using the Lipofectamine 2000 (Invitrogen, USA) following the manufacturer's instructions.

2.3. Isolation and Identification of BMSC-Derived Exosomes

The BMSCs from C57BL/6J mice were isolated as described previously [25]. The protocol of this study was approved by the animal ethic committee of our hospital. The BMSCs were cultured in a low-glucose DMEM (Hyclone, USA) containing 10% FBS (Gibco, USA). The positive and negative cell surface biomarkers including CD29 (65191, 1 : 100, Proteintech, China), CD73 (65162, 1 : 100, Proteintech, China), CD90 (66766, 1 : 100, Proteintech, China), CD105 (67075, 1 : 100, Proteintech, China), CD14 (60253, 1 : 100, Proteintech, China), CD34 (60180, 1 : 100, Proteintech, China), and CD45 (60287, 1 : 100, Proteintech, China) on BMSCs were detected by flow cytometry (BD Biosciences, USA). The exosomes were extracted from the culture medium of BMSCs using an ultracentrifugation method following previous research [26]. Transmission electron microscopy (TEM, Talos L120C G2, FEI, Czech) was adopted to observe the morphology of exosomes. For cellular experiments, exosomes were used at a dose of 1 μg/ml.

2.4. Cell Viability and Apoptosis

The viability of HASMCs and VSMCs was determined by Cell Counting Kit-8 (Biosharp, China) according to the manufacturer's instructions. In brief, cells were seeded onto 96-well plates at a density of 5 × 103 cells/well. Cells were incubated with CCK-8 solution (Biosharp, China) for 1 h. Then, the absorbance values at 450 nm were measured by a microplate reader (Bio-Rad, USA). Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Cells were fixed in 4% paraformaldehyde and stained by in situ cell death detection kit (Roche, USA). The nuclei were dyed with DAPI (Sigma, USA). The positive staining of apoptotic cells was observed by a fluorescence confocal microscope (Leica, Germany).

2.5. Western Blotting

The exosomes and aortic tissues were homogenized by RIPA lysis buffer (Beyotime, China) containing proteinase inhibitors (Sigma, USA) to extract total proteins. The proteins were quantified by the BCA assay kit (Beyotime, China). The proteins were separated by the SDS-PAGE and transferred onto the NC membranes. The membranes were blocked with 1% BSA at 37°C for 1 h. The membranes were incubated with primary antibodies against CD63 (67605, 1 : 5000, Proteintech, China), Hsp70 (66183, 1 : 2000, Proteintech, China), and calnexin (66903, 1 : 5000, Proteintech, China) at 4°C overnight. Subsequently, membranes were incubated with HRP-conjugated anti-mouse secondary antibody (SA00001, 1 : 4000, Proteintech, USA) at room temperature for 45 minutes. The bands were visualized by using an ECL kit (Millipore, Germany) in a gel image system (Bio-Read, USA).

2.6. Real-Time PCR

RNA was isolated from cells, exosomes, and aortic tissues by using the TRIzol reagent (Invitrogen, USA). The cDNA was synthesized by using PrimeScript Master Mix (Takara, Japan). The real-time PCR was performed on the ABI 7500 Real-time PCR system by using the SYBR Premix Ex Taq Kit (Takara, Japan). β-Actin was used as an internal control for normalization of gene expression using the 2- method. The primers were listed as follows:

SBF2-AS1: sense, 5′-CAGAAGGAGUCUACUGCUAAG-3′; antisense, 5′-UAGCAGUAGACUCCUUCUGGG-3′

miRNA-520f-3p: sense, 5′-GTGCCTGTTGCGTCTC-3′; antisense, 5′-GAAAGCCTAGCCGTATTCG-3′

SMARCD1: sense, 5′-ATGACACCTCAGGGACCTTC-3′; antisense, 5′-GGACTGATCCATCCCTGACT-3′

β-Actin: sense, 5′-GAAATCGTGCGTGACATTAA-3′; antisense, 5′-AAGGAAGGCTGGAAGAGTG-3′

U6: sense, 5′-GACCGAGTGTAGCAAGG-3′, antisense; 5′-GTTCTTCCGAGAACATATAC-3′.

2.7. Caspase-3 Activity

The activity of caspase-3 was determined by the Caspase-3 Activity Assay Kit (Thermo, USA). Briefly, HASMCs and VSMCs were suspended in a lysis buffer, and the lysates were mixed with the caspase-3 substrate for 2 h. The absorbance values at 405 nm were determined by a microplate reader (Bio-Rad, USA).

2.8. RNA Pull-Down Assay

The biotin-labeled miRNA-520f-3p and negative control probes were obtained by the Pierce RNA 3′ End Desthiobiotinylation Kit (Thermo, USA). Cells were lysed, sonicated, and incubated with magnetic beads (Invitrogen, USA) conjugated with probes at 4°C overnight. Samples were eluted and the qRT-PCR assay was performed to evaluate the levels of SBF2-AS1.

2.9. Statistical Analysis

Data were presented as mean ± standard deviation (SD), and differences were analyzed by using SPSS 20.0 software (IBM Corp, Armonk, NY, USA). All experiments were performed in triplicate. Differences between two or more groups were determined by independent Student's t-test or one-way analysis of variance (ANOVA) followed by Bonferroni's tests. The two-sided P value less than 0.05 represented statistical significance.

3. Results

3.1. SBF2-AS1 Can Be Transferred to VSMC and HASMC via BMSC-Derived Exosomes

To evaluate the effect of BMSC-derived exosome SBF2-AS1 on the biological function of VSMCs and HASMCs, exosomes were isolated from BMSCs. BMSCs were identified by flow cytometry analysis for positive surface markers (including CD29, CD90, CD105, and CD71) and negative surface markers (including CD14, CD34, and CD45) (Figure 1(a)). Exosomes from BMSCs were successfully observed by TEM and nanoparticle tracking analysis (Figure 1(b)). The expression of exosomal biomarkers CD63 and Hsp70 was detected in exosomes from BMSCs (Figure 1(c)). Furthermore, the expression of SBF2-AS1 was increased or decreased by treating exosomes with BMSCs overexpressing SBF2-AS1 or knocking down SBF2-AS1, respectively (Figures 1(d) and 1(e)), suggesting that SBF2-AS1 can be transferred by BMSC-derived exosomes to VSMCs and HASMCs.

Figure 1

SBF2-AS1 can be transferred by BMSC-derived exosomes to VSMCs and HASMCs. (a) Flow cytometry to detect the positive and negative biomarkers of BMSCs (b). TME to detect the morphology of exosomes. (c) Western blotting assay of biomarkers of exosomes. (d and e) The expression of SBF2-AS1 was detected by qPCR in BMSCs, exosomes derived from BMSCs, VSMCs, and HASMCs after treatment with exosomes derived from BMSCs transfected with overexpressing plasmid or SBF2-AS1 shRNA. All experiments were performed in triplicate. ∗∗ indicated P < 0.01.

3.2. SBF2-AS1 Represses the Survival of VSMCs In Vitro

To assess the effect of BMSC-transferred exosomal SBF2-AS1 on VSMCs, the VSMCs were treated with exosomes derived from BMSCs transfected with overexpressing plasmid or SBF2-AS1 shRNA. The cell viability of VSMCs was enhanced by the depletion of SBF2-AS1 and was reduced by the overexpression of SBF2-AS1 (Figure 2(a)). The silencing of SBF2-AS1 increased and the SBF2-AS1 overexpressing decreased the Edu-positive VSMC numbers (Figures 2(b) and 2(c)). Conversely, the TUNEL-positive VSMC numbers were suppressed by SBF2-AS1 knockdown and were induced by SBF2-AS1 overexpression (Figures 2(d) and 2(e)). Consistently, the caspase-3 activity was inhibited by SBF2-AS1 depletion and was promoted by SBF2-AS1 overexpression in VSMCs (Figure 2(f)).

Figure 2

SBF2-AS1 represses the survival of VSMCs in vitro. (a–f) The VSMCs were treated with exosomes derived from BMSCs transfected with overexpressing plasmid or SBF2-AS1 shRNA. (a) The cell viability was detected by CCK-8 assays. (b and c) The cell proliferation was tested by Edu assays. (d and e) The cell apoptosis was determined by TUNEL assays. (f) The caspase-3 activity was analyzed by the caspase-3 Activity Assay Kit. All experiments were performed in triplicate. ∗∗ indicated P < 0.01.

3.3. SBF2-AS1 Enhances SMARCD1 Expression by Sponging miR-520f-3p in VSMCs and HASMCs

We further investigated the potential binding sites of miR-520f-3p to SBF2-AS1 and SMARCD1 by bioinformatics analysis of the ENCORE database (Figure 3(a)). We validated the overexpression of miR-520f-3p by a miR-520f-3p mimic in VSMCs and HASMCs (Figure 3(b)). The treatment of miR-520f-3p mimic repressed the luciferase activity of SBF2-AS1 and SMARCD1 in VSMCs and HASMCs (Figures 3(c) and 3(d)). RNA pull-down assay revealed the direct interaction of miR-520f-3p with SBF2-AS1 (Figure 3(e)). The depletion of SBF2-AS1 inhibited the expression of SMARCD1, while the inhibitor of miR-520f-3p in VSMCs and HASMCs (Figure 3(f)).

Figure 3

SBF2-AS1 enhances SMARCD1 expression by sponging miR-520f-3p in VSMCs and HASMCs. (a) The potential interaction of miR-520f-3p with SBF2-AS1 and SMARCD1 was predicted by the ENCORE database. (b–d) The VSMCs and HASMCs were treated with miR-520f-3p mimic. The expression of miR-520f-3p was measured by qPCR. (c and d) The luciferase activity of SBF2-AS1 and SMARCD1 was detected by dual-luciferase reporter gene assay. (e) The interaction of SBF2-AS1 and miR-520f-3p was measured by RNA pull-down in VSMCs and HASMCs. (f) The VSMCs and HASMCs were treated with SBF2-AS1 shRNA or cotreated with SBF2-AS1 shRNA and miR-520f-3p inhibitor. The expression of SMARCD1 was determined by qPCR in VSMCs and HASMCs. All experiments were performed in triplicate. ∗∗ indicated P < 0.01.

3.4. SBF2-AS1 Represses the Survival of VSMCs and HASMCs by Targeting miR-520f-3p/SMARCD1 Axis In Vitro

We further validated the function of BMSC-transferred exosomal SBF2-AS1/miR-520f-3p/SMARCD1 signaling in VSMCs and HASMCs, and the VSMCs and HASMCs were treated with exosomes derived from BMSCs transfected with SBF2-AS1 shRNA or cotreated with SMARCD1 overexpressing plasmid or miR-520f-3p inhibitor. The cell viability of VSMCs was enhanced by the depletion of SBF2-AS1 in VSMCs and HASMCs, in which the overexpression of SMARCD1 or the inhibitor of miR-520f-3p could block the effect (Figures 4(a) and 4(b)). Meanwhile, the caspase-3 activity was reduced by the knockdown of SBF2-AS1 in VSMCs and HASMCs, while the SMARCD1 overexpression or miR-520f-3p inhibitor could revere this effect (Figures 4(c) and 4(d)).

Figure 4

SBF2-AS1 represses the survival of VSMCs and HASMCs by targeting miR-520f-3p/SMARCD1 axis in vitro. (a–d) The VSMCs and HASMCs were treated with exosomes derived from BMSCs transfected with SBF2-AS1 shRNA or cotreated with SMARCD1 overexpressing plasmid or miR-520f-3p inhibitor. (c and d) The caspase-3 activity was analyzed by the caspase-3 Activity Assay Kit. All experiments were performed in triplicate. ∗∗ indicated P < 0.01.

4. Discussion

AAA serves as a pathological condition characterized by sustaining dilatation on the aortic wall of proximal thoracic and infrarenal regions, leading to aortic rupture, and bleeding and causing sudden death of patients. BMSCs are multipotent cells and present a remarkable regulatory effect on multiple diseases by delivering exosomes and exosomal lncRNA. In this study, we identified the innovative function of BMSC-derived exosomal SBF2-AS1 in AAA.

BMSCs have been reported to modulate aneurysms in several previous investigations. It has been reported that chemokine receptor-4 from BMSCs regulates abdominal aortic aneurysms and migration of the BMSCs [27]. BMSC-transferred exosomal microRNA-23b-3p regulates T helper/Treg processes via inactivating the PI3k/Akt/NF-κB signaling during aneurysm [28]. Meanwhile, it has been found that exosomal SBF2-AS1 promotes temozolomide chemoresistance of glioblastoma cells [19]. SBF2-AS1 enhances cervical cancer progression by targeting miR-361-5p/FOXM1 signaling [18]. SBF2-AS1 promotes progression and tumorigenesis of breast cancer by regulating microRNA-143/RRS1 axis [21]. SBF2-AS1 induces invasion and proliferation in colorectal cancer by modulating miR-619-5p/HDAC3 expression [29]. SBF2-AS1 regulates the radiosensitivity by targeting the microRNA-302a/MBNL3 axis in the non-small-cell lung cancer [30]. In the present study, we found that SBF2-AS1 could be transferred to VSMCs and HASMCs via BMSC-derived exosomes. The cell viability and proliferation of VSMCs were enhanced by depletion of SBF2-AS1, and overexpression of SBF2-AS1 was able to reduce the proliferation of VSMCs. Conversely, SBF2-AS1 knockdown inhibited apoptosis in VSMCs, whereas SBF2-AS1 overexpression induced apoptosis. Caspase-3 activity was inhibited by depletion of SBF2-AS1 and promoted by overexpression of SBF2-AS1 in VSMCs. These results suggest that BMSC-transferred exosomal SBF2-AS1 contributes to AAA progression and that SBF2-AS1 plays an important role in regulating aneurysms. The clinical role of exosomal SBF2-AS1 transferred from BMSCs in aneurysms needs more studies to confirm in the future.

Furthermore, we found that SBF2-AS1 enhanced the expression of SMARCD1 by forming miR-520f-3p in VSMC and HASMC. Overexpression of SMARCD1 or miR-520f-3p inhibitor reversed cell viability and caspase-3 activity mediated by SBF2-AS1 depletion in VSMC and HASMC. Previous studies have indicated that inhibition of lncRNA SNHG20 repressed the cholangiocarcinoma progression by targeting miR-520f-3p [22]. miR-520f-3p reduces gastric cancer cell proliferation by targeting SOX9/Wnt signaling [31]. SMARCD1 deficiency in VSMCs prevents AAA by inhibiting extracellular matrix degradation and inflammation [28]. Our results suggested a mechanism by which BMSC-transferred exosomal SBF2-AS1 promotes AAA by targeting miR-520f-3p/SMARCD1.

5. Conclusions

Bone marrow mesenchymal stem cell exosome-derived SBF2-AS1 contributes to AAA formation via the miRNA-520f-3p/SMARCD1 axis. Targeting SBF2-AS1 could serve as a promising therapeutic strategy for AAA.