Leptin stimulates the epithelial‑mesenchymal transition and pro‑angiogenic capability of cholangiocarcinoma cells through the miR‑122/PKM2 axis
- Authors:
- Published online on: May 21, 2019 https://doi.org/10.3892/ijo.2019.4807
- Pages: 298-308
Abstract
Introduction
Cholangiocarcinoma, also known as bile duct cancer, is a highly aggressive and metastatic cancer derived from epithelial cells lining the intrahepatic or extrahepatic bile ducts (1). Due to the presence of metastasis at diagnosis for the majority of patients, rendering the tumour inoperable, the overall prognosis for cholangiocarcinoma is poor, and the five-year survival is 5-10% (2). Intensive efforts have been dedicated to developing effective therapies, particularly those targeting the metastatic spread of cholangiocarcinoma.
Cancer metastasis is a multi-step process that involves the local migration and invasion of tumour cells followed by distant dissemination of these cells through blood and lymphatic vessels. Metastatic phenotypes gained by tumour cells, coupled with enhanced angiogenesis, predispose malignant cancer development (3). Epithelial-mesenchymal transition (EMT) is a biological process through which epithelial cells acquire morphological, structural, and functional features of mesenchymal cells, characterized by the downregulation of cell-cell junction components, including E-cadherin and β-catenin, the upregulation of mesenchymal markers, including N-cadherin and catenin, and enhanced migratory and invasive capacities (4,5). EMT has been well demonstrated to be essential for the malignant growth and the metastatic spread of multiple cancer types, including cholangiocarcinoma (6,7). Therefore, characterizing and targeting the molecular mechanisms underlying EMT and angiogenesis may be beneficial for developing treatment strategies against cholangiocarcinoma.
Leptin is an adipocyte-derived cytokine (adipokine) that signals through specific leptin receptors, activates various downstream signalling pathways, and serves multiple roles in human physiology and pathology (8). The well-documented association between obesity and cancer has directed attention towards leptin. Studies over the past decade have reported a positive association between serum leptin levels and multiple cancer types, including breast (9), lung (10), pancreatic (11), and gastrointestinal cancer (12). Furthermore, the signalling mechanisms and functional significance of leptin during the development of multiple cancer types have been demonstrated (13). The primary signal transduction pathways downstream of leptin receptors include Janus kinase 2 (JAK2), phosphoinositide 3-kinase (PI3K), SH2-containing protein tyrosine phosphatase 2/mitogen-activated protein kinase (SHP2/MAPK), 5′-AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC), and mammalian target of rapamycin (mTOR) kinase (14). A recent study on multiple breast cancer cell lines demonstrated that leptin, through the activation of the PI3K/AKT signalling pathway, upregulates pyruvate kinase muscle isozyme M2 (PKM2), a pyruvate kinase isoform specific to embryonic development and absent in the majority of adult tissues (15). PKM2 is overexpressed in breast cancer cells and essential for leptin-induced EMT in these cells (15). Similarly, PKM2 is upregulated in other cancer types, and functionally important for multiple phenotypes, including cancer metabolism, tumour growth and EMT (16,17). In addition to acting on cancer cells, leptin increases the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis of endothelial cells (18). Therefore, leptin is an attractive target for cancer therapy.
Despite the abundant evidence regarding the association between leptin and numerous cancer types, few studies have assessed its significance in cholangiocarcinoma. Fava et al (19) reported that, by activating PI3K/JAK signalling, leptin stimulates the growth and migration of cholangiocarcinoma cells. However, whether leptin is essential for the malignant phenotypes of cholangiocarcinoma and, if so, which mechanisms are responsible remain unclear. To address these questions, the present study focused on EMT and angiogenesis, assessed the significance of leptin in regulating these biological behaviours, and explored the underlying molecular mechanisms.
Materials and methods
Cell culture and treatments
The human cholangiocarcinoma cell lines SK-ChA-1 and TFK-1 and the human umbilical vein endothelial cells (HUVECs) were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Medical Sciences. The SK-ChA-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) and TFK-1 cells were cultured in RPMI-1640 (both from Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% foetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 100 mg/ml streptomycin. HUVECs were cultured in endothelial cell growth medium (PromoCell GmbH). All cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
For leptin treatment, cells in the log phase were treated with leptin (R&D Systems, Inc.) at 0, 50, 100 or 200 ng/ml, for 0, 24 or 48 h. The morphology of cells was imaged under a light microscope (magnification, ×400).
Synthesis and transfection of RNA oligoribonucleotides
Negative control siRNA (si-NC; 5′-GGCCAGACTGGGAAGAAAA-3′), siRNA targeting PKM2 (si-PKM2; 5′-CUUGUCCGAUGUUACCCAATT-3′), miRNA mimics negative control (miR-NC; sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′), miR-122 mimics (sense 5′-UGGAGUGUGACAAUGGUGUUUG-3′ and antisense 5′-AACACCAUUGUCACACUCCAUU-3′), miRNA inhibitor negative control (inhibitor-NC; 5′-CAGUACUUUUGUGUAGUACAA-3′) and inhibitor miR-122 (5′-CAAACACCAUUGUCACACUCCA-3′) were designed and synthesized by Shanghai GenePharma Co., Ltd.
Transfection of siRNA, miRNA mimics or inhibitor was performed using Lipofectamine 2000 reagents (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. In brief, SK-ChA-1 and TFK-1 cells were seeded on 6-well plates (5×105 cells/well) until ~75% confluency was achieved. si-PKM2 (100 nM), miR-122 mimics (50 nM), miR-122 inhibitor (100 nM) or the corresponding scrambled sequence (100 nM; si-NC, miR-NC and inhibitor NC, respectively) that served as negative control were transfected into the cells in serum-free medium using Lipofectamine 2000. At 48 h post-transfection, the levels of PKM2 and miR-122 were quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to evaluate the effect of transfection.
MTT assay
To assess the effects of leptin on cytotoxicity and cell proliferation, TFK-1 cells were seeded into 96-well plates (Corning Incorporated) in triplicate at 5×105 cells/100 μl/well. Upon treating the cells with leptin at 0, 100 or 200 ng/ml for 24 h or 48 h, 20 μl of MTT reagent (5 mg/ml) was added to each well and incubated at 37°C for a further 4 h. Following centrifugation at 250 × g for 5 min at room temperature, the supernatant was discarded from each well. DMSO (150 μl/well) was added to each well to dissolve the formazan crystals. The absorbance was measured using a microplate reader at 490 nm.
Transwell migration and invasion assays
Transwell chambers coated with Matrigel matrix (BD Biosciences) and uncoated Transwell chambers (8-μm pore; Corning Incorporated) were used for the invasion and migration assays, respectively. Briefly, 1×105 SK-ChA-1 or TFK-1 cells were seeded into the top chamber, and 600 μl of medium containing leptin was added to the lower chamber. After 24 h, cells that did not invade through the membrane were removed using a cotton swab. Cells that invaded the membrane and attached to the bottom side of the inserted membrane were stained with 1% crystal violet at room temperature for 10 min, imaged and counted under a light microscope (magnification, ×100).
Extraction of total RNA and RT-qPCR
Total RNA was extracted from SK-ChA-1 or TFK-1 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA was then synthesized with M-MLV reverse transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. Briefly, 1 μg total RNA and 1 μl dNTP mix (10 mM each dATP, dGTP, dCTP and dTTP) were mixed and incubated at 65°C for 5 min. Then, the reaction mixtures, including 4 μl 5X first-strand buffer, 2 μl 0.1 mol DTT and 40 U of ribonuclease inhibitor were incubated at 37°C for 2 min, followed by incubation with 1 μl M-MLV RT at 37°C for 2 min, 50 min at 37°C and heat inactivation at 70°C for 15 min. qPCR was performed using SuperScript® III Platinum® SYBR Green One-Step qRT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) on the ABI PRISM 7300 Fast Real-Time PCR system (Ambion; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. The thermocycling conditions were as follows: Initial 3 min denaturation step at 95°C; followed by 40 cycles at 95°C 3 sec for denaturation; and at 60°C for 30 sec for annealing and extension. Primer sequences are listed in Table I. Each reaction was performed in triplicate, and the relative expression levels of each target gene were calculated as a ratio to that of GAPDH, the internal control, using the 2−∆∆Cq method (20).
Western blot analysis
SK-ChA-1 or TFK-1 cells were collected and lysed using cell lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Subsequently, 30 μg total protein/lane were boiled at 95-100°C for 5 min and separated on a 12% SDS-PAGE gel, and then proteins were transferred onto a polyvinylidene difluoride membrane. Upon blocking the membrane in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) containing 5% non-fat milk at room temperature for 1 h, the target protein was probed with one of the following primary antibodies at 4°C overnight: Rabbit anti-human E-cadherin, (1:1,000; cat. no. A3044), rabbit anti-human β-catenin (1:1,000; cat. no. A10834), rabbit anti-human N-cadherin (1:1,000; cat. no. A0433), rabbit anti-human vimentin (1:1,000; cat. no. A11952), rabbit anti-human VEGFA (1:1,000; cat. no. A0280), rabbit anti-human angiotensin 1 (Ang1; 1:1,000; cat. no. A945; all from ABclonal Biotech Co., Ltd.), anti-PKM2 (1:1,000; cat. no. 4053; Cell Signaling Technology, Inc.), or rabbit anti-human GAPDH (internal control; 1:5,000, AC001, ABclonal Biotech Co., Ltd.). The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. The signal was developed using the Pierce™ Enhanced Chemiluminescence system (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The signal density was analysed using ImageJ software (version 1.51q; National Institutes of Health), and the relative protein level was calculated as the density ratio of the target protein to GAPDH (internal control).
Tube formation assay
To assess in vitro angiogenesis, conditioned medium was collected from cholangiocarcinoma cells. Briefly, 2×105 cells were seeded into 10-cm tissue culture plates. Following an overnight culture, cells were treated with 0 or 200 ng/ml leptin for 24 h. Following three washes with DMEM, cells were cultured in serum-free DMEM for a further 24 h. The conditioned medium was collected and centrifuged at 2,000 × g for 10 min at 4°C to remove any cell debris. A 24-well plate was coated with Matrigel. Upon gel solidification, HUVECs (1×105 cells/well) were seeded on top of the Matrigel in triplicate, treated with a mixture of conditioned medium:EGM2 medium (volume ratio 2:1), and incubated at 37°C with 5% CO2 for 6 h. Each well was imaged under an Olympus DP71 microscope (Olympus Corporation) at ×100 magnification, and the quantification of branching points was performed using ImageJ software (version 1.51q).
Luciferase reporter assay
starBase (http://starbase.sysu.edu.cn/) and miRDB (http://mirdb.org) software tools were used to identify potential miRNAs that may bind to the 3′-untranslated region (3′-UTR) of the human PKM2 gene (21,22). For the luciferase reporter assay, the 3′-UTR sequence of the PKM2 gene containing the potential miR-122-binding site was cloned into a psiCHECK-2 luciferase reporter plasmid (Promega Corporation) and transfected into TFK-1 cells using Lipofectamine 2000 according to the manufacturer's protocol. Upon treating the cells with vehicle control (PBS only), or transfecting miR-122 mimics or miR-122 inhibitor for 36 h, luciferase activity was detected using the Dual Luciferase Reporter assay system (Promega Corporation) according to the manufacturer's protocol. Renilla luciferase activity was normalized to firefly luciferase activity in cells.
Collection of blood samples and ELISA
The present study was approved by the Institutional Review Board of Hunan Provincial People's Hospital, and written informed consent was obtained from all participants. A total of 15 patients with cholangiocarcinoma admitted to the Hunan Provincial People's Hospital between January 2016 and December 2016 were recruited for the present study. The diagnosis for cholangiocarcinoma was established by combining clinical symptoms and examination with ultrasound, computed tomography, and magnetic resonance imaging. Fifteen sex- and age-matched healthy individuals were also recruited as controls. Venous blood was collected from each participant (total 15 patients), and the serum concentration of leptin (cat. no. RAB0333; Sigma-Aldrich; Merck KGaA) and plasma concentration of PKM2 (cat. no. LS-F29732-1; LifeSpan BioSciences, Inc.) were measured by ELISA kit, following the manufacturer's protocols.
Statistical analysis
All data were analysed using SPSS 13.0 software (SPSS, Inc.) and presented as the mean ± standard deviation. The difference between two groups was analysed using an unpaired two-tailed Student's t-test. One-way analysis of variance was used for comparison among multiple groups and multiple comparisons were further performed using the post hoc Tukey's test. P≤0.05 was considered to indicate a statistically significant difference.
Results
Leptin induces EMT in cholangiocarcinoma cells
Accumulating evidence suggests that leptin is a pleiotropic cytokine involved in cancer development (23,24). However, in cholangiocarcinoma, few studies have investigated the functions or molecular mechanisms of leptin (19). To examine the biological significance of leptin, its biosafety and effect on the proliferation of the human TFK-1 cholangiocarcinoma cell line was first assessed using an MTT assay. As shown in Fig. 1A, no significant decreases in cell viability were observed when the cells were treated with 100 or 200 ng/ml leptin for 24 h, and leptin at 200 ng/ml promoted TFK-1 cell proliferation when detected at 48 h, suggesting that leptin is not toxic to TFK-1 cells. Next, the effect of leptin on the migratory and invasive behaviours of TFK-1 cells was examined. In response to increasing concentrations of leptin (0, 100 and 200 ng/ml), leptin significantly stimulated the migration and the invasion of cells in a dose-dependent manner, as indicated by Transwell assay (Fig. 1B and C). Monitoring the changes in cell morphology revealed that leptin transformed the cholangiocarcinoma cells from cuboidal shaped cells to elongated spindle fibroblast-like cells (Fig. 1D), resembling the changes associated with EMT. To follow up, the levels of several EMT promoters, including E-cadherin, β-catenin, N-cadherin and vimentin were measured in cell lines following leptin treatment. As shown in Fig. 1E and F, leptin treatment significantly reduced the protein expression of epithelial markers E-cadherin and β-catenin, while enhancing that of mesenchymal markers N-cadherin and vimentin in a dose-dependent manner. Collectively, these data support that leptin promotes EMT in cholangiocarcinoma cells.
Leptin stimulates the pro-angiogenic capability of cholangiocarcinoma cells
To examine the activity of leptin in angiogenesis, TFK-1 cells were treated with 0 or 100 ng/ml leptin and the conditioned medium was collected from these cells. By applying the conditioned medium to HUVECs, monitoring their angiogenesis (Fig. 2A, left), and measuring the number of branching points (Fig. 2A, right), conditioned medium from leptin-treated cholangiocarcinoma cells was demonstrated to induce tubular formation of HUVECs. Concomitantly, leptin significantly increased the protein expression of VEGFA and Ang1 (Fig. 2B), which are known pro-angiogenic factors in cholangiocarcinoma cells, suggesting that leptin stimulates the pro-angiogenic capability of cholangiocarcinoma cells.
Leptin increases the expression of PKM2 in cholangiocarcinoma cells
To explore the molecular mechanisms underlying the EMT- and angiogenesis-stimulating activities of leptin, PKM2, a pyruvate kinase essential for leptin-induced EMT in breast cancer, was examined (15). By measuring PKM2 on the steady-state mRNA and the protein level in TFK-1 and sk-chA-1 cells, leptin was revealed to significantly upregulate the expression of PKM2 in a dose-dependent manner (Fig. 3A). The time-course study revealed that PKM2 mRNA and protein (Fig. 3B) peaked at 24 h after leptin treatment, and gradually reduced thereafter in TFK-1 and sk-chA-1 cells. Taken together, these results suggest that leptin increased the expression of PKM2 in cholangiocarcinoma cells.
PKM2 is essential for leptin-induced EMT and pro-angiogenesis
To assess the significance of upregulated PKM2 in leptin-mediated EMT or pro-angiogenesis of cholangiocarcinoma cells, the expression of endogenous PKM2 was targeted with siRNA (si-PKM2). RT-qPCR and western blot analysis demonstrated that si-PKM2 significantly and specifically knocked down the level of PKM2 in TFK-1 cells (Fig. 4A). When compared with vehicle control (PBS only) treatment, leptin alone or leptin together with si-NC, si-PKM2 completely abolished the effects of leptin on cell migration (Fig. 4B), invasion (Fig. 4C), and mesenchymal morphology (Fig. 4D). Then, the expression of EMT markers E-cadherin, β-catenin, N-cadherin, and vimentin were detected by western blot analysis, which demonstrated that si-PKM2 abolished the effects of leptin (Fig. 4E). Furthermore, conditioned medium from si-PKM2-treated cholangiocarcinoma cells abolished tubular formation of HUVECs (Fig. 4F) and inhibited the expression of VEGFA or Ang1 (Fig. 4G) in cholangiocarcinoma cells. Taken together, these data suggest that PKM2 is an essential signalling molecule for leptin-induced EMT and pro-angiogenesis of cholangiocarcinoma cells.
miR-122 mediates the regulation of leptin on PKM2
To identify the molecules controlling leptin-mediated regulation on PKM2, a bioinformatics search was performed, and miR-122 was predicted as a potential miRNA that interacts with the 3′-UTR of PKM2 (Fig. 5A). The luciferase reporter assay demonstrated that miR-122 mimics significantly reduced the luciferase activity of the reporter carrying the 3′-UTR of PKM2. In contrast, a mutated luciferase reporter was developed by site-directed mutagenesis, and the results suggested that mutations on the binding sites successfully abolished the suppressive effect of miR-122 (Fig. 5B). Next, miR-122 mimics and miR-122 inhibitors were used for gain-of-function and loss-of-function experiments, respectively. RT-qPCR analysis revealed that miR-122 mimics elevated the miR-122 level to ~23-fold of that in cells transfected with miR-NC, while miR-122 inhibitor significantly reduced the level of miR-122 when compared with the expression in cells transfected with inhibitor NC (Fig. 5C). Consistently, miR-122 mimics significantly reduced, while miR-122 inhibitors potently increased the endogenous mRNA (Fig. 5D) as well as the protein level of PKM2 (Fig. 5E) in cholangiocarcinoma cells. The addition of leptin reduced the inhibitory effect of miR-122 mimics on PKM2 expression (Fig. 5D and E). Furthermore, leptin significantly reduced the miR-122 level in a dose-dependent manner (Fig. 5F). Collectively, these data suggest that leptin may target miR-122, thus releasing miR-122-mediated inhibition on PKM2.
miR-122 mimics reverse leptin-induced EMT and are pro-angiogenesis
To analyse the functional significance of miR-122 as a negative regulator for leptin-mediated expression of PKM2, cholangiocarcinoma cells were treated with leptin together with miR-122 mimics. As shown in Fig. 6A and B, miR-122 mimics were sufficient to reduce leptin-induced migration and invasion of cholangiocarcinoma cells to the basal level (when cells were treated with PBS) and a level significantly lower compared with that in cells treated with leptin alone or leptin with vehicle control (leptin+NC). In addition, miR-122 mimics significantly suppressed the changes in cell morphology (Fig. 6C) and gene expression (Fig. 6D) associated with leptin-induced EMT. The conditioned medium collected from cholangiocarcinoma cells treated with leptin+miR-122 mimics presented similar activities as the medium from cells treated with leptin+si-PKM2 (Fig. 5E), disrupting tubular formation of HUVECs (Fig. 6E), which was associated with the reduced expression of VEGFA and Ang1 (Fig. 6F). Taken together, these results suggested that increasing miR-122 activity is sufficient to reverse leptin-induced EMT as well as the pro-angiogenic capability of cholangiocarcinoma cells.
Serum level of leptin is positively correlated with PKM2, but negatively correlated with miR-122 in cholangiocarcinoma patients
The data presented above support the essential role of the leptin/miR-122/PKM2 axis in regulating EMT and angiogenesis of cholangiocarcinoma. To analyze the significance of this axis in the clinical setting, the serum levels of leptin and miR-122, and plasma level of PKM2 from patients with cholangiocarcinoma and age- and sex-matched healthy individuals were measured. As shown in Fig. 7A-C, serum leptin and PKM2 levels were significantly higher, while that of miR-122 was significantly reduced in patients when compared with the corresponding levels in healthy controls. Further correlation analysis revealed that in the patients, the serum level of leptin positively correlated with that of PKM2, but negatively with that of miR-122 (Fig. 7D).
Discussion
In the present study, it was demonstrated that leptin is a potent cytokine capable of inducing EMT and stimulating the pro-angiogenic capability of cholangiocarcinoma cells. Furthermore, the effects of leptin on cholangiocarcinoma cells were indicated to be mediated through the leptin/miR-122/PKM2 axis as the upregulation of miR-122 or downregulation of PKM2 was sufficient to inhibit leptin-induced phenotypes. Consistently, the serum level of leptin was significantly higher in patients with cholangiocarcinoma compared with healthy controls, and positively correlated with serum PKM2 levels, but negatively correlated with serum miR-122 levels. The results of the present study support the notion that leptin is an ideal therapeutic target for cholangiocarcinoma since antagonizing leptin was able to simultaneously target multiple phenotypes responsible for the malignant progression of the disease.
An increasing body of evidence supports the association between obesity and a higher risk of developing different types of cancer (25,26). In a study on 30 patients with colorectal, breast, lung, testicular or gastric cancer, no significant differences were observed in the serum leptin level at baseline or in response to chemotherapy between patients and matched healthy controls, although leptin was identified to be positively associated with the body mass index of patients (27). By contrast, when focusing on a specific type of cancer, several studies suggest a positive association between higher serum leptin level and cancers (28,29), while some suggest a negative correlation (11,30). Meta-analysis revealed that obesity is a risk factor for cholangiocarcinoma (31). Consistently, the results of the present study demonstrated that the baseline level of serum leptin was significantly higher in cholangiocarcinoma patients compared with that in healthy individuals, indicating that leptin is functionally important for the development of cholangiocarcinoma.
Aggressive development and early metastatic spread constitute the major obstacles for effective treatment of cholangiocarcinoma. Therefore, understanding and targeting mechanisms responsible for the malignancy of the disease may aid in developing novel therapies for cholangiocarcinoma. Previous studies have presented convincing evidence that EMT is present in cholangiocarcinoma development and progression, but also associated with poor prognosis of patients (32,33). In the present study, leptin was revealed to be functionally capable of inducing EMT phenotypes in cholangiocarcinoma cells: Stimulating their migration and invasion; changing tumour cells from the cuboidal epithelial morphology to a spindle mesenchymal shape; downregulating junction molecules, E-cadherin and β-catenin; and upregulating mesenchymal markers, N-catenin and vimentin. Although, to the best of our knowledge, this is the first study to demonstrate that leptin is capable of inducing EMT in cholangiocarcinoma, EMT is not a novel functional phenotype for leptin, as has been well documented in multiple cancer types, including breast (34), lung (35), and ovarian (36) cancer. Several signalling pathways have been identified to mediate leptin-induced EMT, including PI3K/AKT/PKM2 (15), TGF-β (35), AKT/GSK3β and MTA1/Wnt1 axes (34). In the present study, leptin increased the expression of PKM2 in cholangiocarcinoma cells, and siRNA-mediated downregulation of PKM2 was sufficient to abolish multiple leptin-induced EMT phenotypes.
Notably, through bioinformatic miRNA screening, miR-122 was identified as a negative regulator between leptin and PKM2, whereby leptin, through the suppression of miR-122, reversed the inhibitory effects of miR-122 on PKM2 in cholangiocarcinoma cells. The essential role of miR-122 in leptin-PKM2 regulation was further corroborated by the findings that miR-122 mimics were sufficient to downregulate endogenous PKM2, while miR-122 inhibitors significantly increased the expression of PKM2. Karakatsanis et al (37) reported the downregulation of miR-122 in intrahepatic cholangiocarcinoma. Wu et al (38) demonstrated that, by inhibiting p53 expression, miR-122 was necessary and sufficient to reduce the proliferation, and suppress the migration and invasion of cholangiocarcinoma cells. Although these studies support the idea that miR-122 functions as a tumour suppressor, they did not explore the mechanism by which miR-122 inhibited the aggressive and metastatic behaviours, specifically EMT of cholangiocarcinoma.
In addition to tumour cells, other mesenchymal components within the tumour microenvironment, including endothelial cells, fibroblasts, and macrophages are also essential for the malignant progression of cholangiocarcinoma (39). Studies have established leptin as a potent angiogenic factor and revealed multiple underlying mechanisms: Activating P38 MAPK/AKT/COX-2 signalling or JAK/STAT and AKT signalling pathways; and increasing the production of VEGF, fibroblast growth factor-2 (FGF-2) and matrix metalloproteinases from endothelial cells (40). In the present study, the leptin/miR-122/PKM2 axis was demonstrated to regulate the pro-angiogenic capability of cholangiocarcinoma cells in vitro. Conditioned medium from tumour cells treated with miR-122 mimics and si-PKM2 presented comparable effects in disrupting tubular formation associated with the downregulation of VEGF and Ang1 expression in cholangiocarcinoma cells. Although Wang et al (41) suggested that miR-122 directly targeted VEGF through post-transcriptional regulation, the findings of the present study indicate that it may indirectly target VEGF expression via PKM2. Consistently, Azoitei et al (42) demonstrated that PKM2 promoted HIF-1α accumulation and stimulated the subsequent secretion of VEGF from pancreatic cancer cells by activating NF-κB. Li et al (43) reported that PKM2 increased endothelial cell proliferation, migration and adhesion to the extracellular matrix. As the angiogenesis assays were performed on endothelial cells using the conditioned medium from cholangiocarcinoma cells in the present study, the possibility that the leptin/miR-122/PKM2 axis may directly act on endothelial cells to stimulate angiogenesis in an autocrine manner cannot be excluded.
In conclusion, the results of the present study provided clinical relevance and demonstrated the pleiotropic activities of leptin in promoting the malignant transformation of cholangiocarcinoma. Leptin not only stimulates multiple EMT phenotypes, but also enhances the pro-angiogenic activity of cancer cells. Therefore, targeting leptin may aid in supressing a multitude of malignant behaviours of cholangiocarcinoma and achieve superior anticancer effects. Molecularly, the actions of leptin in cholangiocarcinoma cells appear to be mediated through the miR-122/PKM2 axis, allowing for the development of novel anticancer therapies that enable the fine-tuning of leptin activities. As this study was primarily performed using cells cultured in vitro, it is essential to follow up this study with properly established in vivo cholangiocarcinoma models.
Funding
The present study was supported by the Scientific Research Project of Hunan Provincial Education Department (grant no. 17C0963), the Research Foundation of Hunan Provincial Health Bureau (grant no. B2007136) and the Science and Technology Program of Hunan Provincial Science and Technology Department (grant no. 2009SK3079).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
BJ substantially contributed to the conception of this study, as well as edited the manuscript. CP was involved in the study conception and design. ZS performed the literature research and experimental studies. WY performed the clinical studies. OL performed data acquisition and edited the manuscript. ZT and BJ performed data and statistical analysis. CG was involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Institutional Review Board of Hunan Provincial People's Hospital, and written informed consent was obtained from all participants.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Not applicable.
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