Ethics statement

All tissues were obtained with informed consent from the patients. Our study followed the Declaration of Helsinki, and ethical approval was obtained from the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. All animal experiments were carried out approval of the Animal Ethics Committee of The First Affiliated Hospital of Chongqing Medical University, and the experimental process followed the Guidelines for the Use of Laboratory Animals (National Research Council (US) 2011).

Clinical sample collection

A total of 52 pairs of tumor samples and corresponding adjacent non-tumoral tissues of HCCA patients, which were collected from tumor surgery in The First Affiliated Hospital of Chongqing Medical University, were included in this study. Fresh tissue was harvested immediately after surgery and immediately stored in liquid nitrogen after washing with frozen phosphate-buffered saline (PBS).

Cell culture

Human intrahepatic biliary epithelial cells (HIBEpiC) and cholangiocarcinoma cell lines (KKU-100, QBC939, HUCCT1, RBE, HCC-9810) were purchased from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (Invitrogen, Waltham, MA, USA) with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 mg/ mL) (Invitrogen) in a humidified incubator at 37 °C and 5% CO2.

Cell treatment

Cells were transfected with miR-182-5p inhibitors (200 nM/well) (RiboBio, Guangzhou, Guangdong, China), si-FBXW7 (200 nM/well) (RiboBio), and the negative control (NC) (RiboBio) using Lipofectamine 3000 (L3000015, Invitrogen) according to the manufacturer’s instructions. Next, the cells were cultured in a constant temperature incubator for 48 h for subsequent experiments. To obtain cells with stable low expression of miR-182-5p, we packaged antagomiR-182-5p (RiboBio) with adenovirus (RiboBio), which was then infected with KKU-100 cells. We chose stably expressing cells by adding puromycin (2 µg/mL) and used them for the in vivo experiments.

Cell counting kit-8 (CCK-8) assay

The transfected cells were seeded into 96-well plates with 3 × 103 cells/well and incubated at 37℃ for 48 h. After that, 10 µL CCK-8 reagent was added to each well and incubated at 37℃ for 1 h. The absorbance at 450 nm was detected using a microplate reader.

Colony formation assay

Cells were seeded in 6-well plates (500 cells/well) and incubated in RPMI-1640 medium supplemented with 10% FBS for 14 days. The colonies were fixed with methanol and stained with 0.5% crystal violet. Colonies were then counted by two investigators blinded to the study.

Transwell assay

Transwell assay was performed using a 24-well Transwell coated with Matrigel (Corning, New York, USA). In brief, 100 µL of serum-free RPMI 1640 medium containing 106 cells was added to the upper chamber, and 500 µL of RPMI 1640 medium containing 10% FBS was added to the lower chamber. The cells were incubated at 37 °C for 48 h and stained with crystal violet. The number of invasive cells was counted.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells using a TRIzol reagent (Invitrogen). After assessing the quality and concentration of RNA, reverse transcription of RNA into complementary DNA (cDNA) was performed using RevertAid™ (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using Power SYBR Green PCR MasterMix (Thermo Fisher Scientific) on an Applied Biosystems StepOnePlus Real-time PCR system (Thermo Fisher Scientific). The relative expression levels of genes or miRNAs were analyzed by the 2−ΔΔCt method (Livak and Schmittgen 2001). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference for FBXW7, and U6 as the internal reference for miR-182-5p (Zhang et al. 2020b). Primer sequences are shown in Table 1.

Table 1 PCR primer sequencesWestern blot assay

The cells were treated with radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), and the total protein concentration was determined using a bicinchoninic acid kit (Pierce Biotechnology, Rockford, IL, USA). The target protein was isolated by 8–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes. After being blocked with 5% bovine serum albumin for 1 h, the membranes were incubated with primary antibodies against FBXW7 (1:1000, ab192328, Abcam, Cambridge, MA, USA) and β-actin (1:1000, ab8227, Abcam) at 4℃ overnight. After washing with Tris-buffered saline with Tween, the membranes were incubated with the secondary antibodies for 1 h. The signals were detected with an enhanced chemiluminescence detection reagent (Beyotime).

Bioinformatics

StarBase (https://rnasysu.com/encori/) (Li et al. 2014) database was used to predict the miR-182-5p expression in CHOL. StarBase, TargetScan (http://www.targetscan.org/) (Agarwal et al. 2015), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) (Sticht et al. 2018), miRDB (http://mirdb.org/) (Chen and Wang 2020), and miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php) (Huang et al. 2022) were used to predict downstream target genes of miR-182-5p.

Dual-luciferase report assay

The FBXW7 3’UTR sequence binding to miR-182-5p was cloned into the pmirGlo dual-luciferase miRNA target expression vector (Promega, Madison, WI, USA) to construct a luciferase reporter vector (FBXW7-WT), and the sequence containing the binding site with miR-182-5p was mutated to construct a mutant luciferase reporter vector (FBXW7-MUT). The cells were seeded into 96-well plates and transfected with miR-182-5p mimic or mimic NC when they reached 50-70% confluence. The luciferase activity was measured at 48 h after transfection by dual-luciferase reporter kits (Promega).

Nude mice xenograft model

Male BALB/c thymic nude mice (aged 6–8 weeks) purchased from the National Laboratory Animal Center (Beijing, China) were kept under specific pathogen-free conditions for 1 week before the experiment. Cells (4 × 106) with stable low miR-182-5p expression or negative control were injected subcutaneously into the right side of each mouse, and tumor volume was measured every 3 days after the tumor was identified. The formula was calculated: volume (mm3) = length × width2/2. On day 21 after injection, all mice were euthanized by intraperitoneal injection of pentobarbital (200 mg/kg). The tumors were isolated and weighed, and samples of 6 mice in each group were randomly selected for rapid freeze in liquid nitrogen, while the tumors of other 6 mice were used for paraffin embedding and immunohistochemical detection.

Immunohistochemistry (IHC)

Paraffin embedded tissue samples were cut into 5 μm sections. Sections were incubated with Ki67 (1:200, ab16667, Abcam) overnight at 4℃. After washing with PBS, the sections were incubated with secondary antibody immunoglobulin G (1:2000, ab150077, Abcam) at 25℃ for 20 min and then developed by diaminobenzidine. The images were photographed under a microscope (Olympus, Tokyo, Japan).

Statistical analysis

SPSS21.0 statistical software (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, USA, USA) were used for data analyses and data plotting. First, normality and homogeneity of variance tests were conducted, which verified that the data were in normal distribution and homogeneity of variance. Data comparisons between two groups were analyzed by t test; data comparisons among multiple groups were analyzed by one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons for post hoc test. p < 0.05 was considered statistically significant.