AS and healthy samples

A total of 28 AS patients and 28 healthy normal controls in this study were obtained from the Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University. The serum samples were collected and stored in -80 °C. The diagnosis of patients with AS was determined on the basis of carotid intima-media thickness (CIMT) of the common carotid artery (CIMT ≥ 1.0 mm) combination with chest radiography, carotid plaques and the actual pathological indicators of the patient, and patients with other clinical conditions were excluded. The clinical characteristics of AS patients and healthy normal controls are listed in Table 1. The work was agreed by the Ethical Committee of the Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University.

Table 1 The clinical characteristics of AS patients and healthy normal controlsCell culture and treatment

VSMCs were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (11,995,065, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; 10,100,147, Gibco) at 37 °C in 5% CO2. For observing the effect of β-glycerophosphate (β-GP) on the expression of circHIPK3, VSMCs were incubated with 10 mM β-GP in different time points for 0, 3, 6, 9, and 12d. For establishing a vascular calcification model, VSMCs were treated with 10 mM β-GP for 12 d as previously described [20].

Cell transfection

Full-length sequences, including circHIPK3, SIRT1 and mitofusin 2 (MFN2), were amplified and cloned into pcDNA3.1 vector to construct circHIPK3, SIRT1 and MFN2 overexpression vectors. The siRNA negative control (si-NC), siSIRT1 and si-peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were purchased from GenePharma (Shanghai, China). The corresponding cell transfections were performed using Lipo6000™ Transfection Reagent (C0526, Beyotime Biotechnology, Shanghai, China).

Alkaline phosphatase (ALP) activity analysis

ALP activities in different groups were measured by ALP Assay Kit (P0321M). Specifically, the cell lysis buffer for Western and IP without inhibitors (P0013Ja) was applied to disrupt the VSMCs, and the supernatant was obtained by centrifuging at 10,000 rpm/min for 10 min. All above reagents were purchased from Beyotime Biotechnology. Finally, the absorbance at 405 nm was measured using a microplate reader, and the ALP activity was calculated according to the definition of enzyme activity.

Alizarin red staining

As previously described [20], Alizarin red staining was performed to detect the mineralized calcium deposits for VSMCs in different treatments. The VSMCs were rinsed by PBS for three times, fixed with 70% ethanol and stained with alizarin red S solution (1%, pH 4.2) (G1452, Solarbio, Shanghai, China) for 1 h. The VSMCs had been washed 3 times with PBS and mineralized calcium deposits were visualized by microscopy (Leica, Wetzlar, Germany).

Western blot

The total proteins of VSMCs in different groups were extracted by radioimmunoprecipitation analysis buffer (RIPA, R0010) supplemented with 0.5% phenylmethanesulfonyl fluoride (PMSF, P0100). The protein concentrations in different groups were quantified by the bicinchoninic acid (BCA) protein assay kit (PC0020). All above reagents were purchased from Solarbio. Then, protein samples (20 µg total proteins) were submitted to 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After treated with 5% nonfat milk in PBS containing 0.05% Tween-20, membranes were hatched with primary antibodies at 4 °C overnight followed by incubated with secondary antibody for 1 h at room temperature. The primary antibodies were purchased from Abcam (Cambridge, MA, USA): anti-RUNX2 (ab76956, 1: 2000), anti-osteoprotegerin (OPG) (ab73400, 1:3000), anti-alpha smooth muscle actin antibody (SMA) (ab265588, 1:2500), anti-smooth muscle protein 22α (SM22α) (ab14106, 1:1000), anti-FUS (ab243880, 1:1000), anti-SIRT1 (ab110304, 1:1000), anti-PGC-1α (ab106814, 1:1000), anti-GAPDH (ab9485, 1:2500) and anti-MFN2 (ab124773, 1:1000). The blots were visualized using an ECL chemiluminescent reagent (P0018FS, Beyotime Biotechnology), and the densitometry of each protein band was quantified by Image J software. The blots were cut prior to hybridization with antibodies, so there are no images showing full length membranes.

Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

The total RNA extraction of tissue samples and cells with different transfections was performed by Total RNA Extraction Reagent (R401-01). The cDNA was obtained according to the protocol of HiScript II (R212-01). PCR amplification was performed using SYBR qPCR Mix (Q711-02/03). All above reagents were purchased from Vazyme (Nanjing, China). CircHIPK3, RUNX2, OPG, SMA, SM22α, SIRT1, PGC-1α and MFN2 expressions were normalized by GAPDH. The primer sequences are displayed in Table 2.

Table 2 Primers in this work are listedELISA

The concentrations of PGC-1α and MFN2 in cell culture supernatant were detected by PGC-1α ELISA Kit and MFN2 ELISA Kit (Shanghai Jianglai industrial Limited By Share Ltd., Shanghai, China) according to kit instructions.

RNA immunoprecipitation (RIP) assay

RNA Binding Protein Immunoprecipitation Assay Kit (KT102-01; Saicheng Biotech Co., Ltd, Guangzhou, China) was applied to carry out RIP assay. Briefly, 4 × 107 VSMCs were lysed and then 100 µL of supernatant was collected to incubate with the magnetic beads. The magnetic beads were previously conjugated with anti-FUS antibodies (1:1000; ab243880; Abcam) and control anti-IgG antibodies. The magnetic beads were eluted and the RNA complex was purified to obtain the RNA for qRT-PCR analysis.

RNA pull-down assay

RNA pull-down assay was performed by RNA pull-down kit (KT103-01; Guangzhou Saicheng Biotech Co., Ltd). In brief, 4 × 107 VSMCs were lysed and centrifuged to collect the supernatant. The magnetic beads (50 µL) were conjugated with biotinylated circHIPK3 or SIRT1 probes and incubated with the supernatant (100 µL) at 4 °C overnight. Next, the beads were eluted from the RNA-protein complex. The FUS protein was detected by western blot assay.

RNA degradation assay

VSMCs in logarithmic phase were collected and incubated with 0.5 µM actinomycin D (Act D) for 0, 3, 6, 9, and 12 h. After incubation, VSMCs in different groups were collected and lysed to measure the SIRT1 mRNA expression by qRT-PCR.

Statistical analysis

All experiments were performed in triplicate, with each independent experiment set 3 times to generate an average value. Data are presented as means ± SD. Results were analyzed with one-way ANOVA and t-test using GraphPad Prism 7.0 and SPSS 19.0 software. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).