Primary culture of ACP cells

6 patients with primary ACP who underwent surgery from August 2017 to February 2020 at the First Affiliated Hospital of Fujian Medical University were enrolled in this stage. 2 male and 4 female patients were enrolled and the average age of the patients at the time of surgery was 43.33 ± 15.45 years old (range 19–65 years). All patients and/or their legal surrogates provided written informed consent for the use of the tissue specimens. The study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University. Primary ACP cells were cultured and identified as previously reported [14]. Briefly, after tumor resection, solid tumor samples were immediately placed into a cube with DMEM medium (Gibco, Grand Island, USA) containing 10% (v/v)fetal calf serum (Life Technologies, Basel, Switzerland) and penicillin/streptomycin. Then the specimen was delivered for primary cell culture. At this stage, the tumors were cut into small pieces of 1 mm diameter and dispersed by treatment with trypsin for 30 min at 37 °C. Specimens were then filtered and the cell suspension was centrifuged at 800 rpm for 5 min. The cells were washed and then cultured at 37 °C in 5% CO2 atmosphere in a keratinocyte medium (Gibco, Grand Island, USA) with 2 × 105 cells/ml. The cells were characterized by immunocytochemistry. Staining for cytokeratin ((CK, dilution 1:100; 85 ZM-0069, ZSGB-BIO, Beijing, China).

In vitro calcification assay

ACP cells were cultured with Bmp2 for 10 days and then stained with 2% Alizarin red (Sigma-Aldrich). Briefly, after the medium was removed, ACP cells were rinsed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Next, the cells were rinsed twice with PBS and stained with a 2% alizarin red (ph 4.2) (Sigma-Aldrich, St. Louis, MO, USA) working solution for 10 min at room temperature. Finally, the cells were washed with PBS three times, and images were collected.

Western blot analysis

Protein was extracted from the fresh ACP surgical specimens by lysing cells with protease inhibitor cocktail (Roche, USA). After measuring the protein content with a Bradford assay, 20 μg of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and incubated overnight with the following antibodies: anti-HAT, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8 (1:1000, Abcam, USA), anti-CBX4, anti-Runx2, anti-histone (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:1000, Proteintech, USA). The specimens were then incubated with secondary antibodies, IRDye800-conjugated anti-rabbit IgG and IRDye680-conjugated anti-mouse IgG (1:15,000, LiCor, USA), for 1 h at room temperature to label the primary antibody. An Odyssey Infrared Image System (LiCor, USA) was used to analyze signal intensities. The densitometry results were first normalized to the density obtained for β-actin or histone and then compared with that of the control to obtain relative fold changes.

Immunohistochemistry (IHC)

Paraffin-embedded tissue sections from 12 patients with pathologically confirmed ACP were obtained from the pathology department of the First Affiliated Hospital of Fujian Medical University. 5 male and 7 female patients were enrolled and the average age of the patients at the time of surgery was 46.42 ± 16.02 years old (range 14–65 years). These patients underwent surgery from July 2017 to February 2020. Eight of the patients had been confirmed to have obvious tumor calcification while the rest of four patients with no tumor calcification by computed tomography (CT) scan, as described in previous reported [15]. The paraffin sections were stained with anti-HDAC3 antibody (1:100, Abcam, USA) as previously reported [16].

miRNA, siRNA, and vector construct transfection

siRNA targeting HDAC3 for knockdown, miR-129-5p, miR-144, miR-181b, miR-181c, miR-195, miR-200b, miR-410 mimics (mimic-129-5p, mimic-144, mimic-181b, mimic-181c, mimic-195, mimic-200b, and mimic-410), miR-181b inhibitor (inh-181b), miR-200b inhibitor (inh-200b), miR-410 inhibitor (inh-410), siHDAC3, si-CBX4 and negative control (NC) oligonucleotides were purchased from RiboBio (Guangzhou, People’s Republic of China) and transfected into cells using Lipofectamine 3000 (Invitrogen, USA) at a concentration of 50 nM. Q-PCR and western blot was used to assay the transfection efficiency. CBX4-expressing vectors with miR-181b binding sites or mutated miR-181b seed sequences in the MCU 3’-untranslated region (MUT) were purchased from Cyagen Biosciences Inc. (Guangzhou, China). The vectors were subcloned into a psiCHECK-2 vector and then transfected into cells using Lipofectamine 3000 (Invitrogen, USA).

Luciferase assays

The, 3’-UTR of CBX4 and the mutated 3’-UTR of CBX4 were amplified and inserted downstream from the stop codon of Renilla luciferase using a psiCHECK-2 vector (Sagene, China). HeLa cells were cultured in 96-well plates and cotransfected with 10 ng of psiCHECK-2-MCU and 5 pmol of mimic-181b or NC. After incubation for 48 h, firefly and Renilla luciferase activity levels were measured using a dual-luciferase reporter assay system (Promega, Madison, WI) [17].

Immunofluorescence staining

Cells were cultured on a confocal petri dish (NEST Biotechnology Co. Ltd. China), fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. The cells were stained with anti-HDAC3 antibody (1:100, Abcam, USA), which had been diluted 1:200 in 5% goat serum, overnight at 4 °C. The cells were subsequently stained with Alexa Fluor 594 (R37119) (1:200 dilution in PBS) (Invitrogen) at room temperature for 1 h, followed by incubation with DAPI (1:1000 dilution in PBS) for 5 min (min). The cells were then examined with a Lecia laser scanning microscope (FV1000, Olympus) at 100× magnification [18].

Bimolecular fluorescence complementation (BiFC) assay

The BiFC assay was performed to further explore the interaction between CBX4 and HDAC3 in vivo according to previously reported [19]. In brief, the coding region of CBX4 was cloned into pBiFC-mCherryN159. The coding regions of HDAC3 cloned into pBiFC-mCherryC160. pBiFC-mCherryN159-CBX4 and pBiFC-mCherryC160-HDAC3 were co-transfected into ACP cells. 48 h post-transfection, the cells were incubated with Hoechst 33258 for nuclear staining and observed under the laser confocal microscope (FV1000, Olympus).

RNA extraction and miRNA analysis

Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA). The quantity of isolated RNA was determined with a NanoDrop ND-2000 spectrophotometer (Nanodrop Technologies, Delaware, USA). Next, 1000 ng of total RNA was reverse transcribed using a TaqMan microRNA reverse transcription kit (ABI, Forest City, CA). The mRNA and miRNA levels were quantified by qRT-PCR using SYBR Green (Roche, USA) and TaqMan assay kits (ABI) with GAPDH and U6 snRNA used as references, respectively, as previously described [20, 21]. The assays were performed on a 7500 FAST instrument (ABI) under standard conditions as recommended by the manufacturer: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A melting curve analysis was then performed. Relative mRNA and miRNA levels were calculated according to the 2-Δcycle threshold method. The sequences of the PCR primers are shown in Table 1.

Table 1 List of qPCR primers for Osterix, OCN, OPN, and ALP

Statistical analysis

Cell culture experiments were repeated a minimum of 3 times. All quantitative xenograft and in vitro assay results are presented as the means ± standard deviation. Statistical analyses were conducted using SPSS version 13.0 software. All statistical tests were 2-sided. Student’s t-test for bar graph was performed. P values < 0.05 were considered to indicate statistical significance.

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