A total of 122 human CCA and paired paracancerous tissue samples were purchased from Shanghai Outdo Biotech Co., Ltd. (cat. no. JS W-11-01; http://www.superchip.com.cn). All tissues were from patients with CCA who underwent surgery, and none of the patients received any chemotherapy or radiation before surgery. IHC staining was performed as previously described in the literature (15). The experimental protocols and the human tissues used were approved (approval no. 20200305) by the Ethics Committee of Clinical Research of The First Affiliated Hospital of Chengdu Medical College (Chengdu, China). Formalin-fixed and paraffin-embedded tissues (4 µm) were deparaffinized in xylene and rehydrated in ethanol. Subsequently, the tissues were incubated with MCM8 primary antibody (1:100; cat. no. PA5-41325; Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for 4 h and an HRP-conjugated secondary antibody (1:400; cat. no. ab6721; Abcam) at room temperature for 1 h. Following incubation, the tissues were stained with 3,3′-diaminobenzidine solution and hematoxylin at room temperature for 10 min. IHC scores were determined by staining percentage and staining intensity as previously described by Haonon et al (15). In addition, the median IHC score was used to verify the expression levels of MCM8 in CCA.

The human CCA cell lines HCCC-9810, RBE and HuCCT1 were obtained from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and cultured at 37°C in RPMI-1640 medium supplemented with 10% FBS, 100 mg/ml streptomycin and 100 IU/ml penicillin (all from Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO₂.

A total of three short hairpin (sh)RNAs specifically targeting MCM8 (shMCM8) were designed for MCM8 knockdown and the sequences were as follows: RNAi-1: 5′-TGGCAATACATCAGGTGTTAA-3′; RNAi-2: 5′-CTGGAATTGTCAAAGTCTCAA-3′; and RNAi-3: 5′-AGGCAGCTGGAATCTTTGATT-3′. The negative control was a scrambled short interfering (si)RNA (shCtrl: 5′-CCGGAGGGATTGACTTAGAGCAAATCTCGAGATTTGCTCTAAGTCAATCCCTTTTTTG-3′). Lentivirus vectors BR-V108 were purchased from Yi Berry Biological Medicine Technology Co., LTD (http://www.bioscienceres.com). and labeled with green fluorescent protein (GFP) and resistance tag (puromycin, 400 ng/ml) for cell screening. Human CCA cell lines HCCC-9810 and RBE were cultured in 6-well plates (2×10⁵ cells/well; Corning, Inc.) at 37°C for 24 h for second-generation lentiviral transduction. Recombinant lentivirus particles (1×10⁷ TU/ml) contained in shMCM8 or shCtrl sequences were used to transduce CCA cell lines HCCC-9810 and RBE (5×10⁵ cells/ml) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) in RPMI-1640 medium with 10% FBS at a multiplicity of infection of 10 for 40 min at 37°C. A total of 72 h after transduction, the expression of GFP in the cells was observed under a fluorescence microscope (magnification, ×200; Olympus Corporation), and the cells were cultured for 24 h for subsequent experiments.

RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) from target cells. Total RNA was reverse transcribed into cDNA using the M-MLV Reverse Transcriptase kit (Promega Corporation), according to the manufacturer's protocol. qPCR was subsequently performed as follows: Initial denaturation at 95°C for 15 min; 40 of cycles of denaturation, annealing and elongation at 55°C for 15 min; and a final extension at 85°C for 2 min using SYBR-Green Master Mix Kit (Vazyme Biotech Co., Ltd.). The relative mRNA expression of MCM8 was quantified with cycle threshold (Ct) values and normalized using the 2^−∆∆Cq method (16). The primer sequences used were as follows: MCM8 upstream, 5′-ATGGCTTTTCTTTGTGCTGC-3′ and downstream, 5′-CCAGTCCATCGTAACTGTGAGA-3′; GAPDH (internal control) upstream, 5′-TGACTTCAACAGCGACACCCA-3′ and downstream, 5′-CACCCTGTTGCTGTAGCCAAA-3′.

Total protein obtained using RIPA lysis buffer (Beyotime, Jiangsu, China) from HCCC-9810 and RBE cells and quantified using a BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.) and 20 µg/lane protein was separated by SDS-PAGE on a 10% gel [30% acrylamide/bis-acrylamide; 1.5 mol/l Tris (pH 8.8); 10% SDS; 10% ammonium persulfate and tetramethylethylenediamine]. The separated proteins were transferred onto PVDF membranes and blocked using TBST solution containing 5% skimmed milk and 0.5 ml Tween-20 at 4°C for 1 h. Immunostaining was performed using target primary antibodies: Anti-MCM8 (1:1,000; cat. no. PA5-41325; Invitrogen), anti-Akt, (1:1,000; product no. 4685; CST), anti-p-Akt (1:1,000; cat. no. BS-5193R; Bioss), anti-MAPK9 (1:1,000; product code ab76125; Abcam), anti-PIK3CA (1:1,000; product code ab40776; Abcam), GAPDH (1:3,000; cat. no. AP0063; Bioworld) at 4°C for 4 h and corresponding secondary antibody goat anti-rabbit (1:3,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 2 h. Protein bands were visualized using the Amersham ECL + TM Western Blot system (Cytiva) and signal intensities were analyzed using ImageJ software version 1.8.0.112 (National Institutes of Health).

After 5 days of transduction, HCCC-9810 and RBE cells were inoculated overnight on 6-well plates at 2,000 cells/well. A total of 20 µl of 5 mg/ml MTT solution (cat. no. JT343; Genview) was added to each well for 4 h. Subsequently, 100 ml/well DMSO was added and OD values at 490 nm were obtained at 24, 48, 72, 96 and 120 h using a microplate reader. The cell viability was calculated as a ratio of the OD of treated cells to the OD of control cells.

After 5 days of transduction, HCCC-9810 and RBE cells were inoculated on 6-well plates at a density of 1,000 cells/well for 8 days. The number of cells in a single clone was greater than 50, and the size between 0.3–1.0 mm. Subsequently, the cells were fixed with 1 ml 4% paraformaldehyde at room temperature for 30–60 min and stained with 500 µl Giemsa for 10–20 min at room temperature. After washing the cells several times with double distilled water, the cells were captured and counted with a camera (Sony Group Corporation).

After 5 days of transduction, HCCC-9810 and RBE cells were inoculated on 6-well plates at a seeding density of 1,000 cells/well for 10 days. The cells were washed with PBS, centrifuged (4°C, 12,000 × g, 1 min) and resuspended. Subsequently, 500 µl of diluted 1X Annexin V binding buffer (cat. no. 88-8007-74; eBioscience; Thermo Fisher Scientific, Inc.) was added and cells were stained with 10 µl Annexin V-APC for 10–15 min at room temperature in the dark. Subsequently, 5 µl of 50 µg/ml PI was used for staining the cells at room temperature for 20 min and 1X binding buffer was added to assess cell apoptosis using the Guava easyCyte 12HT Benchtop Flow Cytometer and GuavaSuite 19.0 Software (Java; both from Luminex Corporation).

Prepared target cell protein was used to investigate the expression levels of apoptosis-related proteins using the human apoptosis antibody array (cat. no. ab134001; Abcam). After the membrane was blocked with array buffer at room temperature for 1 h, prepared HuCCT1 cell protein was incubated with the membrane at 4°C overnight. The array was subsequently washed, and incubated with detection antibodies (1:100; cat. no. ab134001; Abcam) and streptavidin-HRP (cat. no. ab134001; Abcam) at room temperature for 1 h. Protein expression was visualized using the Amersham ECL + TM Western Blot system (Cytiva) and signal intensities were analyzed using ImageJ software version 1.8.0.112.

After 5 days of transduction, HCCC-9810 and RBE cells were inoculated on 6-well plates at a seeding density of 5×10⁴ cells/well. When the cells had reached a confluence of >90%, the culture medium was replaced with fresh medium. The cell layer was scratched using the scratch instrument (cat. no. VP408FH; V&P Scientific, Inc.) and the cells were cultured in serum-free medium. The shadow area in the center of the 6-well plate was used as a reference and the scratch was in the center of the image. According to the preliminary experiments, images were captured at 0, 4, 8, 24 and 48 h using a fluorescence microscope (magnification, ×200; Olympus Corporation) and the cell migration rate of each group was calculated.

A total of five days after transduction, HCCC-9810 and RBE cells were seeded on a 24-well plate (5×10⁴ cells/well) for 24 h. The chamber in the Transwell transfer test kit (Corning, Inc.) was composed of 24-well tissue culture plates and 12-well cell culture inserts. A total of 100 µl of the cell suspension (5×10⁵ cells) was loaded into the upper chamber of the Transwell and the lower chamber of the Transwell was filled with 500 µl culture medium containing 30% FBS for incubation at 37°C for 48 h. The insert contained a polycarbonate membrane with a pore size of 8 µm, and the cells migrated and adhered to the bottom of the polycarbonate membrane. The cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet solution at room temperature for 20 min. The non-migrating cells were removed and migrated cells were stained. Following washing with PBS, 5 fields of view per well were selected randomly under a fluorescence microscope (magnification, ×200; Olympus Corporation), and images were captured for enumeration of the cells.

Mouse experiments were approved by the Ethics Committee of Chengdu Medical College (approval no. 20190213), following the Guidelines and Procedures for Animal Care and Protection (http://www.lascn.net/Item/7281.aspx). A total of 4-week-old female BALB/c nude mice (Shanghai Lingchang Biotechnology Co., Ltd.) with a body weight of 15–25 g were used to construct the pathogen-free xenograft model. Mice were kept in a non-toxic metal cage with a 12-h light/dark cycle, a constant temperature of 20–22°C, humidity at 30–70%, ventilation every 10–20 times/h and food (cat. no. 000521; Shanghai ZeYa; http://www.shzeya.com) and water intake at any time. At 5 days after transduction, 0.2 ml of shMCM8 and shCtrl HuCCT1 suspended with PBS with a cell concentration of 4×10⁶ cells/ml were injected into the left back of each mouse. The weight and volume of tumors was monitored twice a week, starting at 10 days following injection (calculated by π/6 × L × W²; where L was the long diameter and W was the short diameter). At 46 days after injection, mice were anesthetized using 0.7% pentobarbital sodium at a dose of 40 mg/kg. Subsequently, fluorescence was observed under the imaging system (emission wavelength 510 nm; IVIS Spectrum; Perkin Elmer). Mice were sacrificed by intraperitoneal injection of sodium pentobarbital at a dose of 100 mg/kg according to the American Veterinary Medical Association guidelines (http://www.lascn.net/Item/91107.aspx). The tumors were removed, weighed and images were captured and tumor slides were subjected to Ki67 expression detection using the IHC staining method. Tumor specimens of shMCM8 and shCtrl (4 µm) were fixed with formalin for 30 min, dewaxed with xylene for 15 min and rehydrated with 100% alcohol for 10 min. PBS-H₂O₂ with 0.1% Tween-20 were added and the tissues were washed three times with PBS, 5 min each time. Citric acid buffer was added for antigen retrieval, with heating at 120°C for 20 min. The tumor tissues were incubated with the anti-Ki67 (1:200; cat. no. ab16667, Abcam) at 4°C overnight and secondary antibody incubation with HRP goat anti-rabbit IgG (1:400; cat. no. ab6721; Abcam) at room temperature for 2 h. Images were collected with a microscope (magnification, ×200; Olympus Corporation).

Experimental data were presented as the mean ± standard deviation (n=3) and statistical analyses were performed using SPSS 19.0 (IBM Corp.). The relationship between MCM8 expression and tumor characteristics in patients with CCA was analyzed using chi-square test or Fisher's exact test. Data produced using RT-qPCR was analyzed using 2^−∆∆Cq method. The results of the MTT OD 490-nm value were obtained and wound-healing as well as Transwell assays were investigated using the paired two-tailed Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test analysis. The relationship between MCM8 and clinicopathological features of CCA were analyzed using Mann-Whitney U and Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The results of IHC staining revealed a lower signal intensity of MCM8 in paracancerous tissues when compared with CCA tissues, which indicated that MCM8 expression was significantly higher in CCA tissues compared with paracancerous tissues (Table I and Fig. 1A). Moreover, the relationship between MCM8 expression and tumor characteristics in patients with CCA was analyzed using chi-square test or Fisher's exact test. The results indicated that there was no significant association between the expression level of MCM8 and the pathological stage of CCA (Table II). In addition, Pearson's correlation analysis indicated that the higher the expression level of MCM8, the higher the pathological stage of CCA (Table III). Thus, investigating the expression levels of MCM8 may be clinically relevant in predicting the deterioration of CCA. Furthermore, CCA cell models with MCM8 knockdown were constructed to clarify the effects of MCM8 in CCA cells. The results of the RT-qPCR analysis revealed that the knockdown efficiency of MCM8 in the shMCM8-3 group was the highest compared with other groups (Fig. S1). In addition, a ratio of more than 80% of GFP was observed, indicating that the lentiviruses shCtrl and shMCM8 had successfully infected the CCA cells (Fig. 1B). The results of RT-qPCR demonstrated that the MCM8 expression was decreased by 79.3 and 90.7% in HCCC-9810 and RBE cells compared with the shCtrl group, respectively (Fig. 1C). The protein expression level of MCM8 was also decreased in HCCC-9810 and RBE cells infected with lentivirus shMCM8 (Fig. 1D). Consequently, the low shMCM8-mediated expression levels of MCM8 in HCCC-9810 and RBE cells were used for further functional analysis.

As revealed in Fig. 2A, the effect of shCtrl and shMCM8 on the OD 490-nm values of HCCC-9810 and RBE cells was investigated. The results of the present study demonstrated that the level of cell proliferation was weakened in the shMCM8 group compared with the shCtrl group. Additionally, the colony formation assay in HCCC-9810 and RBE cells indicated that the shMCM8 group yielded smaller and fewer colonies compared with the control group (Fig. 2B). In conclusion, CCA cells with reduced MCM8 expression exhibited impaired proliferation.

The level of cell apoptosis was assessed using flow cytometry. The results indicated that the decrease of MCM8 expression greatly enhanced the apoptotic levels of HCCC-9810 and RBE cells (Fig. 3A). Furthermore, expression levels of a number of apoptotic factors associated with apoptotic signaling pathways were analyzed in cells transfected with shCtrl and shMCM8. As revealed in the Fig. 3B, the expression levels of HSP27, IGF-1sR, survivin, sTNF-R1, sTNF-R2, TNF-α, TNF-β and XIAP were significantly downregulated in HCCC-9810 cells. The results of the present study revealed that MCM8 knockdown enhanced the apoptosis of CCA cells and led to the abnormal downregulation of apoptosis-related proteins.

The effect of MCM8 knockdown on the migration ability of CCA cells was preliminarily evaluated using wound-healing and Transwell assays. Compared with the shCtrl group, the decreased expression of MCM8 resulted in a 16.9% reduction in the migration rate of HCCC-9810 cells within 24 h. The migration rate of RBE cells at 0–48 h decreased by 26.7% (Fig. 4A). Additionally, the results of Transwell assay further indicated that the migration ability of HCCC-9810 and RBE cells in the MCM8-knockdown group was inhibited (Fig. 4B). In conclusion, knockdown of MCM8 inhibited the migration of CCA cells. To further investigate the effect of MCM8 on protein expression in downstream pathways, the expression levels of a number of proteins in well-established signaling pathways were detected in HCCC-9810 cells. The results of western blot analysis indicated that the expression level of MAPK9 was upregulated, while the expression levels of phosphorylated Akt, G1/S-specific cyclin D1 (CCND1) and PIK3 catalytic subunit alpha (CA) were downregulated in the shMCM8 group (Fig. 4C). Mechanistically, the knockdown of MCM8 led to abnormalities in the expression levels of proteins in well-established downstream pathways, such as PI3K/Akt, CCND1 and MAPK.

shMCM8 and shCtrl-mediated HuCCT1 cells were subcutaneously injected into nude mice to determine the effect of MCM8 knockdown in mice. Both groups of mice were anesthetized and images were subsequently captured using an imaging system. The results demonstrated that the intensity of bioluminescence in the shMCM8 group was significantly weaker compared with shCtrl group (Fig. 5A). The results indicated that tumor burden was reduced in mice with low MCM8 expression (Fig. 5B and C). Monitoring of the experimental mice for more than a month revealed that the largest tumor weighed 1.33 g, with a volume of 1,399.91 mm³. In addition, mice with MCM8 knockdown developed tumors that were smaller in size. A maximum tumor diameter of 17.23 mm was observed in the shCtrl group and only 11.59 mm in the shMCM8 group (Fig. 5D). The IHC staining displayed in Fig. 5E further demonstrated that the expression of Ki67 was lower in the shMCM8 group compared with the control group. These results indicated that downregulation of MCM8 suppressed tumor formation in vivo.