Introduction

Biliary tract cancer (BTC) is the second most common malignancy of the hepatobiliary system after hepatocellular carcinoma. BTC includes gallbladder cancer and cholangiocarcinoma (CCA), which is further classified into intrahepatic CCA (iCCA), perihilar CCA (pCCA), and distal CCA (dCCA) based on anatomical location1. China has the highest incidence of CCA globally, approximately 40 times higher than in Western countries2. Early clinical features of CCA are often nonspecific, making diagnosis difficult. More than 65% of patients are diagnosed at an advanced stage, losing the opportunity for curative surgical resection3. Consequently, the 5-year survival rate for CCA patients is extremely low, below 10%4. Even among patients eligible for surgical treatment, the high recurrence rate due to vascular, lymphatic, and neural invasion, distant metastases, and resistance to adjuvant therapies limits the 5-year survival rate to less than 10%5. Surgical resection remains the primary potentially curative treatment for CCA. For early-stage perihilar CCA, liver transplantation following neoadjuvant chemoradiotherapy may be an option. For advanced or unresectable cases, radiotherapy, localized therapies, and systemic chemotherapy are the main treatment modalities6. Imaging modalities for CCA diagnosis primarily include magnetic resonance cholangiopancreatography (MRCP) and computed tomography (CT)7. Beyond established risk factors, recent research has increasingly explored the roles of viral and metabolic factors in CCA pathogenesis8. Due to nonspecific early symptoms, a lack of reliable molecular biomarkers for diagnosis and prognosis prediction, and chemotherapy resistance, the overall incidence of CCA has been increasing9. Understanding the pathophysiological mechanisms of CCA and identifying critical molecules involved in its progression is essential for discovering new diagnostic markers and developing novel therapeutic targets.

The KAL protein, also known as anosmin-1, is an extracellular matrix protein active during embryonic development10. It plays crucial roles in various embryonic systems, including the respiratory tract, kidneys, digestive system, and specific brain regions11. KAL protein is involved in neuronal migration and axonal growth and regulates neural cell interactions. In the developing brain, KAL facilitates the growth and migration of olfactory neurons, which migrate from the developing nasal area to the olfactory bulb in the brain’s anterior region, a process essential for olfactory perception12. Additionally, KAL protein contributes to the migration of neurons producing gonadotropin-releasing hormone (GnRH), a hormone crucial for prenatal and pubertal sexual development. GnRH is essential for normal ovarian and testicular function in females and males, respectively13.

In malignancies, studies suggest a tumor-suppressive role for KAL. For instance, Liu et al. reported that KAL expression was significantly reduced in oral squamous cell carcinoma (OSCC) tissues compared to normal oral mucosa. Reduced KAL expression was associated with advanced clinical stages, lymph node metastasis, and poor prognosis, indicating its potential tumor-suppressive role in OSCC progression14. Tanaka et al. highlighted KAL as a candidate tumor suppressor gene in hepatocellular carcinoma (HCC), with its function potentially suppressed by promoter hypermethylation15. However, the role of KAL in CCA remains unclear. However, the direct molecular mechanisms of KAL in tumor initiation and progression remain unclear. Some studies suggest that the role of KAL in tumors may be mediated through the regulation of the FGFR1 signaling pathway16, while others propose that KAL may regulate the invasive ability of tumor cells by influencing epithelial–mesenchymal transition (EMT)17. Although KAL plays important roles in other cancers, its effect on the growth and proliferation of CCA cells is still unknown.

MicroRNAs (miRNAs) are a class of highly conserved small RNAs of approximately 23 nucleotides in length, widely involved in the regulation of cell proliferation, differentiation, apoptosis, and metabolism. They mainly function by binding to specific mRNAs to regulate gene expression18. The types and levels of miRNAs vary with physiological and pathological conditions, and significant differences in miRNA expression exist between cancer patients and healthy individuals. Therefore, miRNAs have potential as diagnostic and therapeutic biomarkers19. miR-21 is a miRNA with critical regulatory roles in tumorigenesis and progression, and it is upregulated in various cancers such as lung cancer, gastric cancer, and esophageal squamous cell carcinoma20. In addition, studies have shown that miR-21 is highly expressed in CCA patients, and its expression correlates with disease stage and prognosis21. miR-21 promotes CCA cell proliferation and growth, and further research has revealed that this effect is mediated through inhibition of PTEN22. PTEN was the first identified tumor suppressor gene with phosphatase activity, and its expression is often downregulated in cancers. Loss of PTEN function has been shown to activate the PI3K/AKT pathway and promote tumor formation23. In CCA as well, blocking the interaction between PTEN and peroxiredoxin 1 results in PTEN oxidation and loss of phosphatase activity, leading to PI3K/AKT pathway activation and ultimately promoting CCA progression and metastasis24. Based on literature review, our research group hypothesizes that KAL may regulate miR-21, thereby affecting the PI3K/AKT pathway and influencing the progression of CCA. To validate this hypothesis, we designed the present study.

Materials and methods

Cell lines and treatments

The RBE cell line (CCA RBE, CL-0191) was purchased from Procell, and the HUCCT1 cell line (CCA HUCCT1, iCell-h316) was purchased from iCell. Both cell lines were cultured in 1640 complete medium containing 10% FBS and 1% P/S at 37 °C in a 5% CO2 incubator. For generating KAL-overexpressing CCA cells, adenoviral vectors containing KAL overexpression constructs (OE-KAL) or empty plasmids (OE-NC) as negative controls were transfected into RBE or HUCCT1 cells. Cells with 30–50% confluency were treated as follows: using 48-well plates, the medium was replaced with 1 mL of complete medium, followed by the addition of virus (MOI = 20) containing either OE-KAL or OE-NC. Polybrene (5 µg/mL) was added, and cells were incubated for 4–6 h. Complete medium was replenished to 1 mL. On the second day post-infection, the virus-containing medium was replaced with fresh complete medium, and the cells were cultured for 48 h before subsequent experiments.

For generating miR-21-overexpressing HUCCT1 cells, serum-free medium (1 mL) was used to replace the existing medium. Two sterile EP tubes were prepared, each containing 125 µL Opti-MEM. To one tube, 5 µL Lipofectamine 3000 was added, and to the other, 5 µL P3000 and 2.5 µg miR-21 mimic or mimic NC were added. After gentle mixing, the tubes were incubated at room temperature for 5 min. The contents of both tubes were combined, incubated for another 15 min at room temperature, and then added to the corresponding wells of a 6-well plate. Cells were incubated for 4 h, after which 1 mL of complete medium containing 20% serum was added. Experiments proceeded 48 h post-transfection.

Animal handling and procedures

Male BALB/C nude mice (4 weeks old) were obtained from Jiangsu Jicui Yaokang Biological Technology Co., Ltd. (SCXK(Su)2023-0009). Mice were housed at 20–26 °C with a humidity range of 40–70% under ad libitum feeding and water access. Log-phase CCA HUCCT1 cells were prepared as a single-cell suspension at a density of 1 × 10⁸ cells/mL. Mice were immobilized, and the right dorsal region was sterilized with iodine. A 1 mL syringe was used to inject 0.2 mL of the cell suspension subcutaneously into the dorsal region near the axilla. Following tumor growth to 80–100 mm³, mice were randomly assigned to different treatment groups and injected intratumorally with 50 µL of lentivirus (1 × 10⁸ TU) three times at 3-day intervals. Tumor dimensions were measured every 3 days to calculate tumor volume. At the experimental endpoint, intraperitoneal injection of 10 ml/kg of a mixture containing Zoletil (6 mg/ml) and Sumianxin II (1.7857 mg/ml) was administered to rapidly induce anesthesia in mice, ensuring that the animals did not experience pain during the procedure and maintaining a high level of safety. The tumors in mice were measured using a vernier caliper to determine the longest diameter (a) and the shortest diameter (b), and the tumor volume was calculated using the formula: a × b × b × 1/2. All methods were performed in accordance with relevant guidelines and regulations and in accordance with ARRIVE guidelines.

Lentivirus construction

Based on the KAL (NM_000216.4) sequence, an overexpression sequence was designed. The target fragment was obtained by PCR amplification and recombined with the linearized VP-CMV-MCS-3flag-EF1-ZsGreen-T2A-PURO vector. After transformation into competent cells, bacterial colonies were identified by colony PCR and verified by sequencing. Upon successful sequencing, bacterial cultures were expanded, and high-purity, endotoxin-free plasmid DNA was extracted and purified. Using the Easyfectin transfection reagent, the lentiviral transfer plasmid carrying the target fragment, together with the packaging helper plasmids LV003-VSVG, LV003-REV, and LV003-GP, were co-transfected into 293T cells. Viral supernatants were collected at 48 h and 72 h post-transfection. High-titer lentiviral stocks were obtained by ultracentrifugation-based concentration and purification.

CCK8 assay

Cells with 80–90% confluency were seeded into 96-well plates after digestion with 0.25% trypsin (containing 0.02% EDTA) and centrifugation at 1000 rpm for 3 min. Cells were resuspended in medium and plated at a 1:3 ratio. Once adherent, the medium was replaced with fresh medium containing 100 µL per well. CCK8 reagent (10 µL) was added to each well, and cells were incubated for 2 h. Absorbance was measured at 450 nm using a microplate reader (WD-2012B, Beijing LiuYi).

Flow cytometry for apoptosis analysis

Cells (1 × 10⁶) were collected, washed twice with PBS, and resuspended in 300 µL of precooled 1× Binding Buffer. Annexin V-FITC (5 µL) and PI (10 µL) were added, and the mixture was incubated at room temperature in the dark for 10 min. An additional 200 µL of precooled 1× Binding Buffer was added before analysis using a NovoCyte™ flow cytometer (NovoCyte 2060R).

Transwell invasion assay

Cells were digested, centrifuged, and resuspended in incomplete medium. A 24-well Transwell chamber was used, with 300 µL of cell suspension (1 × 10⁵ cells) added to the upper chamber and 600 µL of complete medium in the lower chamber. After 24 h, cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 1 h, and photographed. Inner chamber cells were removed using cotton swabs. Stained cells were dissolved in 33% acetic acid, and 200 µL of the solution was measured at 562 nm using a microplate reader.

ELISA for PIP3 quantification

ELISA plates were equilibrated to room temperature, and standard or sample wells were prepared by adding 50 µL of standards or diluted samples (1:5 dilution). HRP-conjugated detection antibodies (50 µL) were added, and the plate was sealed and incubated at 37 °C for 60 min. After washing five times, 50 µL of Substrate A and 50 µL of Substrate B were added. The plate was incubated at 37 °C in the dark for 15 min, followed by the addition of 50 µL of Stop Solution. Optical density was measured at 450 nm within 15 min.

qPCR analysis

RNA was extracted using Trizon reagent (1 mL Trizon with 0.2 mL chloroform). miRNA was purified using a miRNA extraction kit (CW0627S, CWBIO), and concentration and purity were measured via UV spectrophotometry (OD260/OD280). cDNA was synthesized using a miRNA reverse transcription kit (MR101-02, Vazyme). qPCR was performed using a fluorescence-based PCR system (CFX Connect™, Bio-Rad) under the following conditions: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, for 40 cycles. U6 was used as an internal reference, and relative gene expression was calculated using the 2-ΔΔCt method. The primer sequences were listed in the Table 1.

Table 1 Primer Sequences.
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Western blot analysis

Cells were harvested, and the culture medium was discarded. Total protein was extracted on ice using RIPA lysis buffer and centrifuged at 12,000 r/min at 4 °C for 10 min. The supernatant was collected, and protein concentration was determined using a BCA protein quantification kit (E-BC-K318-M, Elabscience). Proteins were denatured and separated via SDS-PAGE for 1 h before being transferred onto PVDF membranes (Millipore). Membranes were blocked with 3% skim milk at room temperature for 1 h. Primary antibodies used included Mouse Anti-β-Actin (HC201, TransGen Biotech, 1/2000), Rabbit Anti-PTEN (22034-1-AP, Proteintech, 1/2000), Rabbit Anti-Cleaved-Caspase-3 (YC0006, Immunoway, 1/1000), Rabbit Anti-AKT (10176-2-AP, Proteintech, 1/1000), Rabbit Anti-P-AKT (AF0016, Affinity, 1/1000), Rabbit Anti-P-MTOR (AF3308, Affinity, 1/1000), Mouse Anti-MTOR (66888-1-Ig, Proteintech, 1/5000), and Rabbit Anti-BCL-2 (YM3041, Immunoway, 1/1000). Membranes were incubated with primary antibodies overnight at 4 °C. The next day, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Secondary antibodies included HRP-conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000) and HRP-conjugated Goat Anti-Rabbit IgG (H + L) (GB23303, Servicebio, 1/2000). PVDF membranes were treated with enhanced chemiluminescence reagent and visualized using an ultra-sensitive chemiluminescence imaging system (Tanon-5200, Shanghai Tanon Technology Co., Ltd.).

Immunohistochemistry

Tumor tissues from nude mice were sectioned, baked, deparaffinized, and rehydrated. Antigen retrieval was performed using citrate buffer. Sections were blocked with 5% BSA and incubated with primary antibodies, including Ki67 (HA721115, HUABIO, 1/200), Bcl-2 (ET1720-53, HUABIO, 1/200), Cleaved-Caspase-3 (25128-1-AP, Proteintech, 1/200), and PTEN (22034-1-AP, Proteintech, 1/200), overnight at 4 °C. Secondary antibody incubation was performed using HRP-conjugated Goat Anti-Rabbit IgG (ZB-2301, ZSGB-Bio, 1:100). DAB chromogen (CW0125M, CWBIO) was applied, followed by counterstaining with hematoxylin (ZLI-9610, ZSGB-Bio). Sections were dehydrated, cleared, and mounted for observation under a microscope (CX43, Olympus).

Dual-luciferase assay

A dual-luciferase reporter vector was constructed. When cell confluence reached approximately 70%, transfection was initiated. Prior to transfection, the culture medium was replaced with serum-free medium. After 6 h of transfection, 0.5 mL of complete medium containing 20% serum was added to each well of a 12-well plate. According to the grouping described above, 293T cells were transfected, and fluorescence intensity was measured using a fluorescence spectrophotometer to verify whether miR-21-5p directly targets and binds to the PTEN gene.

Statistical analysis

GraphPad Prism 9.0 software was used for data visualization and statistical analysis. All experiments were repeated three times, and quantitative data were expressed as mean ± standard deviation (X ± S). One-way ANOVA was used for multiple group comparisons, and T-tests were used for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

Results

KAL inhibits CCA cell proliferation, migration, and promotes apoptosis

First, the efficiency of KAL overexpression in HUCCT1 and RBE cells was validated through qPCR and Western blot analyses. As shown in Fig. 1A, KAL protein expression was significantly elevated in the KAL overexpression group compared to the Control and OE-NC groups, confirming the successful transfection of the KAL overexpression plasmid into HUCCT1 and RBE cells. Subsequently, cells transfected with empty vectors (OE-NC) or KAL overexpression vectors were subjected to CCK8 assays to assess cell proliferation, flow cytometry for apoptosis analysis, and Transwell assays for invasion capabilities. As shown in Fig. 1B; Table 2, the viability of HUCCT1 and RBE cells in the KAL overexpression group was significantly decreased compared with the normal control and empty vector groups. In addition, as shown in Fig. 1C; Table 2, the apoptosis levels of HUCCT1 and RBE cells were significantly increased in the KAL overexpression group compared with the control and empty vector groups. Furthermore, as shown in Fig. 1D; Table 2, the invasion ability of HUCCT1 and RBE cells in the KAL overexpression group was significantly reduced compared with the control and empty vector groups. These results indicate that KAL can inhibit the proliferation and invasion of cholangiocarcinoma (CCA) cells while promoting their apoptosis.

Table 2 Statistical summary of CCA cell phenotypes.
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Fig. 1
figure 1

KAL inhibits CCA cell proliferation and migration while promoting apoptosis. (A) Western blot validation of KAL overexpression plasmid transfection efficiency in HUCCT1 and RBE cells. (B) CCK8 assay measuring proliferation levels of HUCCT1 and RBE cells. (C) Flow cytometry analysis of apoptosis levels in HUCCT1 and RBE cells. (D) Transwell assay evaluating the invasion capabilities of HUCCT1 and RBE cells (*P < 0.05 vs. Control; #P < 0.05 vs. OE-NC).

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KAL downregulates miR-21-5p expression in CCA cells

The expression levels of miR-21-5p in CCA cells were assessed using qPCR. As shown in Fig. 2A and B, the expression of miR-21-5p was significantly reduced in the KAL overexpression group compared to the Control and OE-NC groups in both HUCCT1 and RBE cells. These results indicate that KAL overexpression effectively downregulates miR-21-5p expression.

Fig. 2
figure 2

KAL downregulates miR-21-5p expression in CCA cells. (A) qPCR analysis of miR-21-5p expression in HUCCT1 cells. (B) qPCR analysis of miR-21-5p expression in RBE cells (n ≥ 3; *P < 0.05 vs. Control; #P < 0.05 vs. OE-NC).

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KAL suppresses PIP3 production in CCA cells

The levels of PIP3 in HUCCT1 and RBE cells were measured using ELISA. As shown in Fig. 3, PIP3 levels were significantly reduced in the KAL overexpression group compared to the Control group. These results indicate that KAL overexpression effectively suppresses PIP3 production.

Fig. 3
figure 3

KAL suppresses PIP3 production in CCA cells. ELISA analysis of PIP3 levels in HUCCT1 and RBE cells (*P < 0.05 vs. Control; #P < 0.05 vs. OE-NC).

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KAL inhibits PTEN/AKT pathway and modulates apoptosis-related protein expression in CCA cells

Western blot analysis was conducted to evaluate the expression levels of PTEN, AKT, p-AKT, MTOR, p-MTOR, Bcl-2, and Cleaved-Caspase3 in HUCCT1 and RBE cells. As shown in Fig. 4A and B, the expression ratios of p-AKT/AKT, p-MTOR/MTOR, and Bcl-2 were significantly reduced in the KAL overexpression group compared to the Control group. Conversely, PTEN and Cleaved-Caspase3 expression levels were significantly increased, with statistical significance. These results indicate that KAL overexpression suppresses AKT phosphorylation, inhibits the AKT signaling pathway, and promotes apoptosis in CCA cells.

Fig. 4
figure 4

KAL inhibits the AKT pathway and modulates apoptosis-related protein expression in CCA cells. (A) Western blot analysis of AKT pathway and apoptosis-related protein expression in HUCCT1 cells. (B) Western blot analysis of AKT pathway and apoptosis-related protein expression in RBE cells (n ≥ 3; *P < 0.05 vs. Control; #P < 0.05 vs. OE-NC).

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Validation of the targeted interaction between PTEN and miR-21-5p

To confirm the targeted interaction between PTEN and miR-21-5p, a dual-luciferase reporter assay was performed. As shown in Fig. 5, the results demonstrate a direct interaction between PTEN and miR-21-5p.

Fig. 5
figure 5

Dual-luciferase reporter assay validating the targeted interaction between PTEN and miR-21-5p (*P < 0.05 vs. PTEN wild-type + mimic NC group; #P < 0.05 vs. PTEN mutant + miR-21-5p mimic).

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Validation of miR-21 mimic transfection efficiency

To investigate the regulatory mechanism of KAL in CCA cells, mimic NC and miR-21 mimic were transfected into HUCCT1 and RBE cells. As shown in Fig. 6, the miRNA expression levels of miR-21-5p were significantly elevated in the miR-21 mimic group compared to the normal control group, confirming the effectiveness of the miR-21 mimic transfection.

Fig. 6
figure 6

qPCR validation of miR-21 mimic transfection efficiency (*P < 0.05 vs. Control; #P < 0.05 vs. mimic NC).

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Table 3 miR-21 reverses the effects of KAL on CCA cell phenotypes.
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miR-21 reverses the effects of KAL on CCA cell proliferation and migration

HUCCT1 and RBE cells with KAL overexpression were transfected with either mimic NC or miR-21 mimic. As shown in Fig. 7A; Table 3, the cell viability of the OE-KAL + miR-21 mimic group was significantly increased compared with the OE-KAL and OE-KAL + mimic NC groups. As shown in Fig. 7B; Table 3, the apoptosis level of the OE-KAL + miR-21 mimic group was significantly decreased compared with the OE-KAL and OE-KAL + mimic NC groups. As shown in Fig. 7C; Table 3, the invasion ability of the OE-KAL + miR-21 mimic group was significantly increased compared with the OE-KAL and OE-KAL + mimic NC groups. These results indicate that miR-21 overexpression can markedly reverse the effects of KAL on the proliferation, apoptosis, and migration of cholangiocarcinoma (CCA) cells.

Fig. 7
figure 7

miR-21 reverses the effects of KAL on CCA cell proliferation, migration, and apoptosis. (A) CCK8 assay for proliferation levels in HUCCT1 and RBE cells. (B) Flow cytometric analysis of apoptosis in CCA cells (C) Transwell assay for invasion levels in HUCCT1 cells (n ≥ 3;*P < 0.05 vs. OE-KAL; #P < 0.05 vs. OE-KAL + mimic NC).

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miR-21 reverses the effects of KAL on PIP3 secretion in CCA cells

As shown in Fig. 8, the PIP3 levels in the OE-KAL + miR-21 mimic group showed an increasing trend compared to the OE-KAL and OE-KAL + mimic NC groups in both cell lines, with statistically significant differences observed in HUCCT1 cells. These results suggest that miR-21 significantly reverses the inhibitory effects of KAL on PIP3 secretion in CCA cells.

Fig. 8
figure 8

miR-21 reverses the effects of KAL on PIP3 secretion in CCA cells. ELISA analysis of PIP3 levels in HUCCT1 and RBE cells (n ≥ 3; *P < 0.05 vs. OE-KAL; #P < 0.05 vs. OE-KAL + mimic NC).

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miR-21 reverses the effects of KAL on the AKT pathway and apoptosis-related protein expression in CCA cells

Western blot analysis was conducted to examine the expression levels of AKT, P-AKT, MTOR, P-MTOR, PTEN, Cleaved-Caspase3, and BCL-2 in HUCCT1 cells. As shown in Fig. 9A, P-AKT/AKT, P-MTOR/MTOR, and BCL-2 protein expression levels were elevated in the OE-KAL + miR-21 mimic group compared to the OE-KAL + mimic NC group. Conversely, PTEN and Cleaved-Caspase3 expression levels were significantly reduced.

Similarly, in RBE cells, as shown in Fig. 9B, P-AKT/AKT, P-MTOR/MTOR, and BCL-2 protein levels were significantly increased in the OE-KAL + miR-21 mimic group compared to the OE-KAL + mimic NC group, while PTEN and Cleaved-Caspase3 levels were significantly decreased. These results indicate that miR-21 can reverse the effects of KAL on the PTEN/AKT pathway and apoptosis-related protein expression in CCA cells.

Fig. 9
figure 9

miR-21 reverses the effects of KAL on the AKT pathway and apoptosis-related protein expression in CCA cells. (A) Western blot analysis of Cleaved-Caspase3, PTEN, AKT, P-AKT, MTOR, P-MTOR, and BCL-2 protein expression levels in HUCCT1 cells. (B) Western blot analysis of Cleaved-Caspase3, PTEN, AKT, P-AKT, MTOR, P-MTOR, and BCL-2 protein expression levels in RBE cells (n ≥ 3; *P < 0.05 vs. OE-KAL; #P < 0.05 vs. OE-KAL + mimic NC).

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In vivo validation of the effects of KAL overexpression lentivirus on CCA tumorigenesis

HUCCT1 cells were subcutaneously injected into nude mice. When tumors reached 80–100 mm³ in size, the mice were divided into groups and treated with the corresponding lentivirus. Tumor weight and volume were recorded in the supplement Table 1. The results are shown in Fig. 10. Figures 10A–B present the viral qPCR and Western blot validation results, showing that KAL expression was significantly increased in the KAL overexpression group compared with the Control and Vector groups. Figure 10C shows the images of tumor formation in nude mice, Fig. 10D illustrates the changes in tumor volume over time, and Fig. 10E presents the statistical analysis of tumor volumes at the experimental endpoint. After injection of HUCCT1 cells into nude mice, the tumor volume gradually increased. However, compared with the Control group, tumor volume decreased progressively after injection of KAL-overexpressing lentivirus. Figure 10F shows the statistical analysis of tumor weight changes, indicating that tumor weight tended to decrease in the KAL overexpression group, although the difference was not statistically significant. These findings suggest that KAL overexpression can inhibit tumor formation of CCA cells in vivo.

Fig. 10
figure 10

In vivo validation of the effect of KAL-overexpressing lentivirus on tumor formation of CCA cells. (A) qPCR verification of viral transfection efficiency; (B) Western blot verification of viral transfection efficiency; (C) Images of tumor formation in nude mice; (D) Changes in tumor volume over time; (E) Statistical analysis of tumor volume at the experimental endpoint; (F) Statistical analysis of tumor weight; n ≥ 3.

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KAL suppresses proliferation and promotes apoptosis in CCA xenografts

Immunohistochemical analysis was performed on tumor tissues to assess the positive expression levels of Bcl-2, Cleaved-Caspase3, PTEN, and KI67. As shown in Fig. 11, compared to the Control group, the KAL overexpression group exhibited significantly increased positive expression of Cleaved-Caspase3 and PTEN, while the positive expression levels of KI67 and Bcl-2 were significantly reduced. These results indicate that KAL overexpression suppresses CCA cell proliferation and promotes apoptosis in CCA xenografts.

Fig. 11
figure 11

KAL suppresses proliferation and promotes apoptosis in CCA xenografts. Immunohistochemical analysis of KI67 and apoptosis-related protein positive expression in tumor tissues. (A) Immunohistochemical staining images. (B) Quantitative analysis of protein positive expression (n ≥ 3; *P < 0.05 vs. OE-KAL; #P < 0.05 vs. OE-KAL + mimic NC).

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Discussion

Cholangiocarcinoma (CCA) is a malignant epithelial tumor of the biliary tract with no specific tumor markers currently identified, making early clinical detection extremely challenging. As a result, most CCA patients are diagnosed at advanced stages, with the disease showing significant resistance to various chemotherapeutic drugs25,26. Research into the molecular mechanisms and signaling pathways underlying CCA’s development, migration, and metastasis remains limited compared to other digestive system tumors. This highlights the urgent need for fundamental research on CCA to reveal its pathogenesis and identify specific tumor markers for early diagnosis27,28.

In this study, we constructed KAL-overexpressing CCA cell lines via adenoviral transfection. Our findings demonstrated that KAL overexpression significantly suppressed CCA cell proliferation and migration while promoting apoptosis. Furthermore, KAL overexpression markedly reduced tumor volume in a mouse xenograft model, accompanied by decreased KI67 expression and increased levels of apoptosis-related proteins, suggesting that KAL may serve as a critical suppressor in CCA progression.

Phosphatidylinositol-3,4,5-triphosphate (PIP3), an essential intracellular second messenger, is generated by the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) via phosphatidylinositol-3-kinase (PI3K)29. PIP3 plays a pivotal role in various cellular processes, including growth, survival, metabolism, migration, and cell cycle regulation. It recruits and activates AKT (protein kinase B) and other PH-domain-containing signaling proteins to the plasma membrane, triggering downstream signaling pathways23. The downstream AKT pathway is crucial in CCA development. In CCA, AKT signaling promotes tumor growth through mTORC1 activation, which facilitates cell proliferation and growth via alternative kinase pathways rather than lipid synthesis, as observed in hepatocellular carcinoma30. Moreover, AKT pathway activation is closely associated with CCA cell proliferation and migration, and its inhibitors can promote apoptosis and suppress tumor growth31,32. Consistent with the findings of Zhang et al. regarding Tip6033, we observed that KAL overexpression suppressed CCA cell proliferation and migration while reducing PIP3 production and AKT pathway activity. This suggests that KAL’s inhibitory effects on CCA progression are closely related to PIP3/AKT pathway suppression.

miR-21 is a non-coding single-stranded RNA molecule comprising approximately 18 to 27 nucleotides. It plays a significant role in tumorigenesis by regulating target gene-associated signaling pathways34. miR-21 is frequently upregulated in various cancers, and its abnormal expression is closely associated with tumor progression. It promotes tumor cell proliferation, inhibits apoptosis, enhances invasiveness and metastasis, and contributes to resistance to chemotherapy and radiotherapy35,36,37. Liu et al. reported significantly elevated miR-21 levels in the serum of CCA patients, correlating with tumor stage, lymph node metastasis, and poor prognosis21. Lu et al. demonstrated that miR-21 promotes CCA progression and tumor growth by targeting NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH)38. Wang et al. further revealed that miR-21 enhances CCA proliferation and growth by inhibiting PTPN14 and PTEN, two functional targets22. In this study, we found that the inhibitory effects of KAL on CCA cell proliferation, migration, and apoptosis, as well as its suppression of the PIP3/AKT pathway, were significantly reversed by miR-21 mimic transfection. These findings suggest that KAL’s inhibitory role in CCA progression is closely linked to miR-21. Our findings on miR-21’s promotion of the AKT pathway in CCA cells are consistent with the results of Liu et al.39.

This study has some limitations. First, the effects of KAL on the AKT pathway were only partially reversed by miR-21 mimic, and some protein expression differences were not statistically significant. This suggests that other regulatory mechanisms may be involved, warranting further investigation. Additionally, the inhibitory effects of KAL on tumor growth in the mouse xenograft model were not statistically significant, likely due to the choice of CCA cell lines. Future studies will focus on selecting CCA cell lines with the lowest KAL expression levels to provide stronger evidence supporting KAL’s role in CCA progression. Moreover, we acknowledge that our current data are based primarily on PTEN expression levels and indirect readouts such as PIP3 reduction. these findings do not fully exclude the possibility that other phosphatases (e.g., SHIP1/2) may also contribute to the observed changes in PIP3 levels. More direct evidence of PTEN activation, such as measurement of its phosphatase activity using commercial activity assays, or analysis of critical phosphorylation sites would provide stronger mechanistic support. Although such assays were beyond the scope of the present work, they represent an important direction for our future studies.

In summary, KAL may suppress cholangiocarcinoma growth by downregulating miR-21 and modulating the PTEN/AKT signaling pathway.