Patient samples

Human CCA tissue samples were obtained from the Department of Biliary and Pancreatic Surgery at Tongji Hospital, Huazhong University of Science and Technology in Wuhan, China. These samples were diagnosed as CCA through pathological examination following surgical resection. Patient follow-up was conducted after surgery, and the date of death or last follow-up was recorded. The procedures for collecting patient tissue samples were ethically approved by the Party Committee Technology of Tongji Hospital, Huazhong University of Science and Technology, and adhered to the principles of the Declaration of Helsinki.

Cell culture

Human CCA cells (TFK1, QBC939, EGI1, HuCCT1, HuH28, RBE, SSP25, HCCC9810) were cultured in RPMI 1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (NEWZERUM, New Zealand), 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were incubated in a cell culture incubator at 37 °C with 5% CO2. QBC939 and HCCC9810 cells were treated with 5 μM or 10 μM decitabine (DAC) (#S1200, Selleck Chemicals, Shanghai, China) for 72 h or 96 h with daily change of complete medium.

Plasmid construction

The full-length sequences of DNMT1, DNMT3a, DNMT3b, DNMT3l, TET1, TET2, and TET3 were amplified by PCR from human cell cDNAs and cloned into pHAGE-Flag vector. Gene-specific sequence small hairpin RNA primers, provided by Sangon Biotech, were inserted into the pLKO.1 vector. The target methylation or target demethylation plasmids, sgRNA-dCas9DNMT3a and sgRNA-dCas9TET1CD, were constructed by recombination of PCR-amplified DNMT3a sequence and TET1CD sequence into PX458-dCas9. The sgRNA targeting RSPO3 was inserted into dCas9DNMT3a and sgRNA-dCas9TET1CD after BbsI enzymatic digestion of the plasmid. The empty vector sgRNA-dCas9DNMT3a was used as a control for targeted methylated control experiments, while the empty vector sgRNA-dCas9TET1CD was used as a control for demethylated experiments. The primers used are shown in Additional file 1.

Viral packaging and viral infection

The target gene plasmid was co-transfected with pMD2G and psPAX packaging plasmids in HEK293T cells. The cells were cultured in DMEM high glucose medium (HyClone, USA) supplemented with 10% fetal bovine serum (NEWZERUM, New Zealand). After 72 h, the supernatants were collected using a 0.45 μm filter (BS-PES-45, Biosharp, China). TFK1 and QBC939 cells were then treated with 1 ml of viral supernatant and 1 ml of RPMI 1640 complete medium, along with 2 μl of polybrene (40804ES76, Yeasen, China), for 24 h. Subsequently, the cells were screened with puromycin hydrochloride (CL13900, Selleck, USA) at a concentration of 1 μg/ml for 2 weeks. Finally, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to assess the efficiency of target gene knockdown or expression.

Cell proliferation assay

Cell viability was assessed using the Cell Counting Kit (CCK-8) (40203ES92, Yeasen, China). A total of 1000 cells were plated into 96-well plates and incubated at 37 °C with 5% CO2. CCK-8 reagent was then added to the wells following the manufacturer's instructions. Absorbance at 450 nm was measured using the Thermo Scientific™ Multiskan™ FC instrument (Thermo Fisher, Shanghai, China). For colony formation assays, 1000 cells were grown in 6-well plates with 2 ml of fresh medium and incubated for 2 weeks. Visible colonies were stained with 0.5% crystal violet, and the number of colonies was counted.

Total RNA isolation and reverse transcription real-time PCR

Total RNA was extracted using the RNA isolator Total RNA extraction reagent (TRIzol, Vazyme, Nanjing, China). cDNA was extracted using HiScript®III RT SuperMix for RT-qPCR (Vazyme) following the manufacturer's protocol. ChamQ Universal SYBR RT-qPCR Master Mix RT-qPCR (Vazyme) was utilized, with β-actin serving as the internal reference. The analysis of the results was performed using Bio-Rad CFX Manager 2.1. Additional file 1 provides information about the primers used.

Bisulfite sequencing PCR (BSP)

BSP primers were designed using the online MethPrimer program (http://www.urogene.org/methprimer). Primers information is found in Additional file 1. DNA extraction was performed using a DNA extraction kit (D3396020000J12T006, Omega, USA), and the BSP transformation reaction was carried out using a kit (EM101-02, Vazyme, China). PCR amplification was then conducted using Taq enzyme (C601-02, Vazyme, China). The PCR products were purified for vector ligation (EM101-02, Vazyme, China), and a total of 10 bacterial clones were randomly selected for sequencing.

Immunohistochemical (IHC) staining

Tumor tissue was embedded in paraffin and sectioned to prevent desiccation. The sections were then baked at 70 °C for 2 h. Deparaffinization with xylene and hydration with gradient ethanol were carried out. Antigen repair was performed by treating the sections with sodium citrate solution in boiling water for 20 min. To prevent nonspecific binding, a 5% BSA solution was applied for 30 min. The sections were then incubated with the primary antibody at 4 °C for 12 h. Nuclei were visualized using diaminobenzidine for 1 min and stained with hematoxylin. Differentiation was achieved by treating the sections with hydrochloric acid in ethanol, followed by dehydration with gradient ethanol, rinsing with xylene, and air drying. The sections were sealed with neutral adhesive and observed under a light microscope. Images were captured for analysis. The number of cancer cells expressing the target protein was counted, and correlation analysis was performed.

Experiments with animals

BALB/c (nu/nu) male nude mice and male C57BL/6J mice were obtained from Jiangsu GemPharmatech and housed in a specific pathogen-free facility at the Animal Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. For xenograft experiments, 5-week-old male BALB/c (nu/nu) nude mice were randomly assigned to groups (n = 8 per group). 3 × 106 cells were suspended in 100 μl PBS and injected subcutaneously into the dorsal side of the mice. Tumor measurements were recorded weekly, and the tumor volume was calculated using the formula: total tumor volume (mm3) = 0.52 × length × width × width. After 5 weeks, the mice were euthanized by dislocating the cervical vertebrae, and the tumors were surgically removed for further analysis.

A primary CCA mouse model was created by diluting 20 μg of pT3-EF1aH-myr-Akt and 20 μg of pT3-EF1aH-NICD1 together with 6 μg of transposon plasmid pCMV(CAT)T7-SB100 in 2.0 mL of saline. The resulting plasmid mixture (2 mL) was injected into the lateral tail vein of 5-week-old male C57BL/6J mice within 7 s. To achieve RSPO3 knockdown, the plasmid pT3-EF1aH was converted into pT3-U6, and a small hairpin RNA specific for the Rspo3 gene was designed and ligated into the pT3-U6 vector. An additional 20 μg of pT3-U6-Rspo3#1, pT3-U6-Rspo3#2 plasmid, or the corresponding control plasmid pT3-U6 was added to the above plasmid mixture. To overexpress Rspo3, the pT3-EF1aH-Rspo3 plasmid was constructed using pT3-EF1aH. The pT3-EF1aH-Rspo3 plasmid or the corresponding control plasmid pT3-EF1aH was added to the above plasmid mixture and injected into the lateral caudal vein of male C57BL/6J wild-type mice. Livers were collected 4 or 5 weeks after hydrodynamic transfection for analysis of CCA tumorigenesis and progression.

Statistical analysis

All experiments were conducted independently, with each experiment repeated at least three times. The data are presented as the mean ± standard deviation, unless otherwise specified. Statistical analysis was performed using GraphPad Prism software 8.0 (GraphPad Prism Software Inc., San Diego, CA, USA). The Student's t-test was used to compare two independent groups. Survival curves were generated using the Kaplan–Meier method and analyzed using the log-rank test. Statistical significance was defined as p values < 0.05. The levels of significance were indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.