Use of slug inhibitors in the preparation of therapeutic drugs for clonorchis sinensis extracellular vesicle-induced cholangiocarcinoma

Abstract

The application discloses application of a Slug inhibitor in preparation of a therapeutic drug for cholangiocarcinoma induced by Clonorchis sinensis extracellular vesicles, and also includes application in preparation of an inhibitor for epithelial-mesenchymal transition, migration, proliferation and invasion of cholangiocarcinoma cells induced by Clonorchis sinensis.The application further provides a drug for treating cholangiocarcinoma induced by Clonorchis sinensis, which comprises siRNA with Slug gene as a target.The application firstly proves that Clonorchis sinensis extracellular vesicles promote proliferation, migration, invasion and epithelial-mesenchymal transition of cholangiocarcinoma cells, and knocking down Slug in the cholangiocarcinoma cells can inhibit the progress of the physiological activities, thereby providing a new technical option for clinical treatment of cholangiocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of cancer treatment, and more specifically, to the use of Slug inhibitors in the preparation of therapeutic drugs for cholangiocarcinoma induced by Clonorchis sinensis extracellular vesicles. Background Technology

[0002] Clonorchiasis, also known as liver fluke disease, is a parasitic disease caused by the parasite *Clonorchis sinensis*, which infects the bile ducts of humans and other mammals. Infection is commonly caused by consuming raw or undercooked freshwater fish or shrimp containing *Clonorchis sinensis* metacercariae. Adult *Clonorchis sinensis* worms primarily parasitize and lay eggs within the human bile ducts, causing hepatobiliary disorders characterized by hepatobiliary epithelial hyperplasia, bile duct structural disorder, and bile duct dilation. Common complications include cholecystitis, cholangitis, and gallstones. Clonorchis sinensis infection is closely related to the development of bile duct cancer, and the International Agency for Research on Cancer (IARC) has classified *Clonorchis sinensis* as a Group 1 carcinogen.

[0003] Cholangiocarcinoma (CCA) is a malignant tumor originating from the epithelial cells of the bile ducts. Based on anatomical location, it can be divided into intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, and distal cholangiocarcinoma. Its onset is insidious, with no obvious clinical symptoms in the early stages, but the disease progresses rapidly, is highly invasive, and is prone to recurrence and metastasis, making radical resection difficult, thus resulting in a poor prognosis for patients. The causes of cholangiocarcinoma are complex and vary geographically. In areas where liver flukes are not endemic, primary sclerosing cholangitis, congenital bile duct diseases, intrahepatic bile duct stones, cirrhosis, non-alcoholic fatty liver disease, and viral hepatitis are important risk factors for cholangiocarcinoma. However, in areas endemic to liver flukes, especially in Southeast Asia where *Clonorchis sinensis* and *Opisthorchis vivierrini* are prevalent, the risk of cholangiocarcinoma is significantly increased. The exact mechanism by which Clonorchis sinensis causes cholangiocarcinoma remains to be elucidated. It is likely the result of the synergistic effects of multiple factors, including mechanical stimulation by the parasite, secondary inflammatory response, toxic effects of secreted / excreted products, abnormal host immune response, and molecular and gene mutations.

[0004] Extracellular vesicles (EVs) are a type of vesicle-like body secreted by various living cells, containing many important bioactive substances such as proteins, lipids, and nucleic acids, participating in mediating important physiological processes such as intercellular communication and immune regulation. In the pathogenesis of Clonorchis sinensis, Clonorchis sinensis extracellular vesicles (CsEVs) can be endocytosed by host bile duct cells, thereby affecting the activation of host immune cells and the expression of related immune factors, playing a crucial role in long-distance communication between the host and the worm. However, the role of CsEVs in Clonorchis sinensis promoting the malignant phenotype of cholangiocarcinoma remains unclear. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides the application of Slug inhibitors in the preparation of therapeutic drugs for cholangiocarcinoma induced by Clonorchis sinensis extracellular vesicles.

[0006] The first objective of this invention is to provide an agent for inhibiting the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of a therapeutic drug for Clonorchis sinensis-induced cholangiocarcinoma.

[0007] A second objective of this invention is to provide an agent for inhibiting the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of an inhibitor of epithelial-mesenchymal transition induced by Clonorchis sinensis in cholangiocarcinoma cells.

[0008] A third objective of this invention is to provide an agent for inhibiting the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells and its application in the preparation of an inhibitor of Clonorchis sinensis-induced migration of cholangiocarcinoma cells.

[0009] A fourth objective of this invention is to provide an agent for inhibiting the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells and its application in the preparation of an inhibitor of Clonorchis sinensis-induced cholangiocarcinoma cell proliferation.

[0010] The fifth objective of this invention is to provide an agent for inhibiting the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells and its application in the preparation of an inhibitor of Clonorchis sinensis-induced invasion of cholangiocarcinoma cells.

[0011] The sixth object of the present invention is to provide a drug for treating cholangiocarcinoma induced by Clonorchis sinensis.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] This invention uses ultracentrifugation to isolate and purify cytotoxic endothelial cells (CsEVs) from the culture supernatant of adult Clonorchis sinensis. Through experiments including CCK8 assay, EdU-488 assay, colony formation assay, flow cytometry, cell cycle analysis, cell scratch assay, and transwell assay, it was confirmed that CsEVs affect the proliferation, migration, and invasion of cholangiocarcinoma cell lines RBE and HuCCT1. The expression levels of epithelial-mesenchymal transition (EMT)-related marker molecules were identified using RT-qPCR and Western blot. Furthermore, this invention uses LC-MS / MS proteomic analysis to identify parasite-derived proteins in CsEVs, aiming to reveal the role and specific molecular mechanism of CsEVs in promoting the malignant phenotype of cholangiocarcinoma in Clonorchis sinensis.

[0014] Therefore, the present invention claims protection for the following:

[0015] The application of an agent that inhibits the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of a therapeutic drug for Clonorchis sinensis-induced cholangiocarcinoma.

[0016] Application of a reagent that inhibits the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of an inhibitor of epithelial-mesenchymal transition induced by Clonorchis sinensis in cholangiocarcinoma cells.

[0017] Application of a reagent that inhibits the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of an inhibitor of Clonorchis sinensis-induced migration of cholangiocarcinoma cells.

[0018] Application of a reagent that inhibits the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of an inhibitor of Clonorchis sinensis-induced cholangiocarcinoma cell proliferation.

[0019] Application of a reagent that inhibits the expression of the Slug gene and / or Slug protein in cholangiocarcinoma cells in the preparation of an inhibitor of Clonorchis sinensis-induced invasion of cholangiocarcinoma cells.

[0020] Preferably, the reagent comprises siRNA targeting the Slug gene. The NCBI number of the Slug gene is 6591.

[0021] More preferably, the nucleotide sequence of the target sequence of the siRNA is shown in SEQ ID NO.13.

[0022] More preferably, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.14, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.15.

[0023] This invention also claims a drug for treating cholangiocarcinoma induced by extracellular vesicles of Clonorchis sinensis, the drug comprising siRNA targeting the Slug gene, the nucleotide sequence of the sense strand of the siRNA being shown in SEQ ID NO. 14, and the nucleotide sequence of the antisense strand being shown in SEQ ID NO. 15. The NCBI number of the Slug gene is 6591, and the nucleotide sequence of the target sequence of the siRNA is shown in SEQ ID NO. 13.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention is the first to demonstrate that extracellular vesicles of Clonorchis sinensis promote the proliferation, migration, invasion, and epithelial-mesenchymal transition of cholangiocarcinoma cells, and that knocking down Slug in cholangiocarcinoma cells can inhibit the process of the above physiological activities, providing a new technical option for the clinical treatment of cholangiocarcinoma. Attached Figure Description

[0026] Figure 1 Results of isolation, purification and identification of extracellular vesicles of Clonorchis sinensis; A: Culture of adult Clonorchis sinensis; B: Isolation and purification of extracellular vesicles by ultracentrifugation; C: Morphological identification of extracellular vesicles of adult Clonorchis sinensis by negative staining transmission electron microscopy; D: Identification of diameter and particle size distribution of extracellular vesicles of adult Clonorchis sinensis by Nanosight.

[0027] Figure 2 A shows the proteomic analysis results of extracellular vesicles of Clonorchis sinensis; B shows the subcellular localization of CsEVs protein; C shows the GO analysis of CsEVs protein; D shows the KEGG analysis results of CsEVs protein (Top 20); and D shows the signaling pathway distribution of CsEVs protein (Top 20).

[0028] Figure 3 This study investigated the endocytosis of extracellular vesicles of Clonorchis sinensis by cholangiocarcinoma cells.

[0029] Figure 4 Results of extracellular vesicles of Clonorchis sinensis promoting the proliferation of cholangiocarcinoma cells; A: CCK8 assay to detect the proliferative effect of different concentrations of CsEVs; B: EdU-488 assay to detect the proliferative effect of CsEVs, with PBS as a negative control, ****P<0.0001; C: Plate colony formation assay to detect the proliferative effect of CsEVs, with PBS as a negative control, RBE: *P<0.05, HuCCT1: ***P<0.001; D: Flow cytometry to detect cell cycle distribution, with PBS as a negative control, PI=(S+G2 / M) / (G0 / G1+S+G2 / M), ****P<0.0001.

[0030] Figure 5 The results show that extracellular vesicles of Clonorchis sinensis promote the proliferation of cholangiocarcinoma cells by regulating cell cycle-related molecules; A shows the expression level of cell cycle-related regulatory factor mRNA in RBE cells by RT-qPCR, with PBS as a negative control; B shows the expression level of cell cycle-related regulatory factor protein in RBE cells by Western blot; C shows the expression level of cell cycle-related regulatory factor mRNA in HuCCT1 cells by RT-qPCR, with PBS as a negative control; D shows the expression level of cell cycle-related regulatory factor protein in HuCCT1 cells by Western blot. **P<0.01, ***P<0.001, ****P<0.0001.

[0031] Figure 6 The results show that extracellular vesicles of Clonorchis sinensis promote the migration and invasion of cholangiocarcinoma cells; A is the cell scratch assay to detect the effect of CsEVs on the migration of cholangiocarcinoma cells, with the PBS group as a negative control, ****P<0.0001; B is the transwell assay to detect the effect of CsEVs on the migration and invasion of cholangiocarcinoma cells, with the PBS group as a negative control, ****P<0.0001.

[0032] Figure 7 Western blot results of extracellular vesicles from Clonorchis sinensis inducing EMT in cholangiocarcinoma cells were obtained, and the protein expression levels of EMT-related marker molecules and transcription factors were detected.

[0033] Figure 8 The results show that Slug plays an important role in CsEVs-mediated EMT and cell migration and invasion; A is the protein expression level of EMT-related markers and transcription factors after Slug knockdown detected by Western blot; B is the Slug gene expression level detected by RT-qPCR after Slug knockdown; C is the effect of Slug knockdown on the migration and invasion of cholangiocarcinoma cells detected by transwell assay; siCon is the Slug knockdown negative control group, and siSlug is the Slug knockdown group. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0035] Example 1: Isolation, purification, and identification of extracellular vesicles (CsEVs) of Clonorchis sinensis

[0036] I. Experimental Methods

[0037] 1. Collection, culture, and collection of culture supernatant of adult Clonorchis sinensis.

[0038] Anesthetize wildcats infected with Clonorchis sinensis and dissect their livers. Using manual finger pressure, squeeze the adult Clonorchis sinensis worms from the edge of the liver towards the common bile duct. Gently pick up clusters or single worms and place them in sterile PBS containing 10% v / v antibiotics (100 mg / L penicillin and 100 mg / L streptomycin). Rinse the worms twice more with sterile PBS containing 10% v / v antibiotics. Figure 1 As shown in A in the diagram.

[0039] Add 2 mL of DMEM medium containing 5% v / v antibiotics to each well of a 6-well cell culture plate. Transfer 20–30 adult Clonorchis sinensis worms into each well and incubate at 37°C with 5% CO2. Change the medium and collect the culture supernatant daily for a total of 3 days.

[0040] 2. Isolation and purification of extracellular vesicles from Clonorchis sinensis

[0041] Extraction was performed using ultracentrifugation, a method widely used in extracellular vesicle research. The procedure is as follows: Figure 1 As shown in B, the specific steps are as follows:

[0042] The culture supernatant of adult Clonorchis sinensis obtained in the previous step was centrifuged at 1500×g for 30 min at 4℃ to remove the precipitate (cell debris and impurities), and the supernatant was collected. It was then centrifuged at 3500×g for 30 min at 4℃ to remove the precipitate (cell debris and impurities), and the supernatant was collected. It was then centrifuged at 12000×g for 30 min at 4℃ to remove the precipitate (apoptotic bodies), and the supernatant was collected. It was then centrifuged at 20000×g for 60 min at 4℃ to remove the precipitate (microvesicles), and the supernatant was collected and filtered through a 0.22 μm filter. It was then centrifuged at 120000×g for 70 min at 4℃ to collect the precipitate (i.e., CsEVs). The precipitate was resuspended in PBS, centrifuged at 120000×g for 70 min at 4℃ to further purify the CsEVs, and the precipitate (i.e., CsEVs) was collected and resuspended in PBS to obtain the CsEVs suspension.

[0043] The protein concentration of CsEVs suspension was determined using the BCA method.

[0044] 3. Identification of extracellular vesicles of Clonorchis sinensis

[0045] (1) Transmission electron microscopy (TEM) was used to identify the size and morphology of CsEVs.

[0046] Take 20 μL of CsEVs suspension and drop it onto a copper mesh carbon support film with a pore size of 120 μm. Incubate at room temperature (25 °C) for 10 min. Blot away excess liquid with filter paper, add 10 μL of 3% v / v phosphotungstic acid staining solution for negative staining for 1–2 min. Blot away excess liquid with filter paper, then wash twice with deionized water. Blot away excess liquid with filter paper, place the copper mesh on the sample holder of a transmission electron microscope, and observe and acquire images using an 80 kV electron beam.

[0047] (2) Nanoparticle tracking analysis (NTA) technique for analyzing CsEV particle size

[0048] Slowly inject ddH2O into the sample chamber using a 1 mL syringe, and place it on a nanoparticle tracer analyzer for detection and zeroing. Take 10 μL of CsEVs suspension, dilute it with ddH2O at a ratio of 1:100 to 1000 μL, inject it into a clean sample chamber, and place it on a nanoparticle tracer analyzer for detection. Perform 3 biological replicates. Export the data from the computer and statistically analyze the particle size and distribution of CsEVs.

[0049] II. Experimental Results

[0050] like Figure 1 As shown in C, the extracellular vesicles isolated from the culture supernatant of adult Clonorchis sinensis have a typical saucer-like structure, and their morphology, structure, and size (30nm–150nm) are similar to those reported in the literature. Figure 1 As shown in D, the main peak value of extracellular vesicles of adult Clonorchis sinensis is 72 nm, and the diameter range is mainly distributed between 30 nm and 150 nm.

[0051] The above results demonstrate that this embodiment successfully isolated and purified Clonorchis sinensis extracellular vesicles (CsEVs).

[0052] Example 2: Extracellular vesicle protein profile and bioinformatics analysis of Clonorchis sinensis

[0053] I. Experimental Methods

[0054] 1. Protein extraction

[0055] Add 100 μL of protein lysis buffer (add protease inhibitor before experiment, manufacturer: Sangon Biotech, catalog number: C600386-0001) to 100 μL of CsEVs suspension prepared in Example 1, sonicate on ice 3 times (sonicate for 2 seconds, stop for 2 seconds, sonicate for 1 min, 5% power), place on ice for lysis for 30 min, then centrifuge at 17000×g, 4℃ for 15 min, and collect the supernatant for later use.

[0056] 2. Enzymatic hydrolysis

[0057] Take the supernatant obtained in the previous step, add dithiothreitol (DTT) reagent to a final concentration of 50 mM, and react at 37°C for 1 hour; then add iodoacetamide (IAM) reagent to a final concentration of 100 mM, and react at room temperature (25°C) in the dark for 40 min to obtain the reduced alkylated protein solution; transfer to a 10 kDa ultrafiltration tube, centrifuge at 12000 × g for 20 min, and discard the solution at the bottom of the collection tube; add 100 μL of 8 M urea (pH 8.5), centrifuge at 12000 × g for 20 min, discard the solution at the bottom of the collection tube, and repeat twice; add 25 mM carbon... Centrifuge 100 μL of ammonium bicarbonate solution at 12000×g for 20 min, discard the solution at the bottom of the collection tube, and repeat 3 times. Replace with a new collection tube, add 50 μL of 25 mM ammonium bicarbonate solution (containing trypsin, trypsin to protein ratio of 1:50) to the ultrafiltration tube, and react at 37℃ overnight (15 h). The next day, add trypsin (trypsin to protein ratio of 1:100), react at 37℃ for 4 hours, centrifuge at 12000×g for 20 min, and collect the solution at the bottom of the tube (i.e., the peptide solution after enzymatic digestion). Add 50 μL of 25 mM ammonium bicarbonate solution to the ultrafiltration tube, centrifuge again at 12000×g for 20 min, collect the solution at the bottom of the tube, and combine it with the bottom solution obtained in the previous step to obtain a total of 100 μL of enzymatically digested peptide solution. Add 100 μL of 0.2% v / v trifluoroacetic acid (TFA) solution to the enzymatically digested peptide solution, mix well, and obtain the peptide sample.

[0058] After activating the C18 column with acetonitrile (ACN), wash twice with 1 mL of 0.1% v / v TFA solution; transfer the peptide sample to the C18 column and discard the dripping liquid; wash again with 500 μL of 0.1% v / v TFA solution and discard the dripping liquid; elute with 500 μL of 70% v / v ACN solution, collect the liquid, and vacuum dry to obtain the peptide sample.

[0059] 3. ZipTip C18 desalination

[0060] Rinse the tip 10 times with 50 μL of 60% v / v ACN / 0.1% v / v TFA; wash the tip 10 times with 10 μL of 0.1% v / v TFA. Aspirate the sample and expel the tip, repeating 20 times, then expel the liquid; wash the tip 5 times with 10 μL of 0.1% v / v TFA. Elute with 10 μL of 60% v / v ACN, collect the eluted peptide sample into a new EP tube, and vacuum dry.

[0061] 4. LC-MS / MS liquid chromatography-mass spectrometry detection

[0062] The peptide sample obtained in the previous step was dissolved in 20 μL of dissolving solution (water containing 0.1% v / v formic acid and 5% v / v acetonitrile), vortexed thoroughly, centrifuged at 13500×g at 4℃ for 20 min, the supernatant was collected and transferred to a sample tube, and 8 μL was taken for LC-MS / MS liquid chromatography-mass spectrometry identification.

[0063] 5. Database Analysis

[0064] The database was searched using PEAKS software, with the following specific search parameters:

[0065] Using the uniprot database: uniprot-Proteome ID_UP000286415_20221115.fasta; trypsin digestion, allowing a maximum of 3 missed cleavage sites, fixed modification as Carbamidomethylation (C), dynamic modification as Oxidation (M) and Acethyl (Protein N-term); maximum error of precursor ion is 15 ppm, maximum error of fragment ion is 0.05 Da; based on peptide identification results, FDR≤0.05 (High Confident) is used for screening and output of results.

[0066] 6. Bioinformatics Analysis

[0067] The subcellular localization of CsEVs proteins identified by mass spectrometry was predicted using WoLF PSORT software; CsEVs proteins were analyzed from the perspectives of biological processes, cellular components, and molecular functions using the online GeneOntology (GO) analysis tool; and KEGG metabolic pathway analysis was performed on CsEVs proteins.

[0068] II. Experimental Results

[0069] LC-MS / MS analysis identified 114 proteins, of which 11 were uncharacterized. Comparison revealed that CsEVs contained heat shock protein 90, GAPDH, and other proteins most commonly identified in extracellular vesicles, as well as proteins frequently identified in parasitic EVs.

[0070] like Figure 2 As shown in A, the subcellular localization results indicate that CsEVs proteins are mainly cytoplasmic proteins, with the remainder being nuclear and plasma membrane proteins. Figure 2 As shown in B, CsEVs proteins are mainly involved in metabolic processes; in terms of cellular components, they are mainly involved in the cytoskeleton and nucleosomes; and in terms of molecular function, the main annotation entry is binding. For example... Figure 2 As shown in C and D, the CsEVs proteins identified by KEGG analysis are involved in 91 signaling pathways, including gap junctions, phototransduction, bile secretion, and bacterial infection, with most of the proteins involved in metabolic pathways.

[0071] Example 3: The role of extracellular vesicles of Clonorchis sinensis in promoting the malignant phenotype of cholangiocarcinoma.

[0072] I. Experimental Methods

[0073] 1. Extracellular vesicle endocytosis experiment of Clonorchis sinensis

[0074] (1) Staining

[0075] The CsEVs prepared in Example 1 were resuspended in PKH26 dye working solution (200 μL Diluent C + 1 μL PKH26) and bound in the dark for 5 min. Then, an equal volume of PBS containing 1% v / v BSA was added to stop the staining, thus obtaining PKH26-labeled CsEVs.

[0076] Dissolve PKH26-labeled CsEVs in PBS, centrifuge at 120,000×g for 70 min to wash, discard the supernatant, and repeat the washing once; resuspend PKH26-labeled CsEVs in 200 μL of sterile PBS and use them directly for subsequent experiments or store them briefly at 4°C.

[0077] (2) Co-cultivation

[0078] Cell crawling smears and RPMI-1640 medium containing 10% v / v fetal bovine serum and 1% v / v penicillin antibiotics were pre-added to 6-well cell culture plates, and cultured at a rate of 5 × 10⁶ cells / well. 5 RBE cholangiocarcinoma cells were added to each well. After the cells grew into a monolayer, PKH26-labeled CsEVs obtained in the previous step were added, with PKH26-PBS set up as a negative control. The cells were incubated at 37°C in a 5% CO2 cell culture incubator in the dark for 3 h, 6 h, and 12 h.

[0079] (3) Laser confocal detection

[0080] Discard the cell supernatant and wash the bile duct cancer cells three times with warm PBS; add 4% v / v paraformaldehyde (Beyotime, LOT: P0099) and fix at room temperature (25℃) for 15 min; add PBS containing 0.3% v / v Triton X-100 for permeation for 10 min; add PBS containing 1% v / v BSA for blocking for 20 min; wash three times with PBS; let the cells air dry naturally, pick up the coverslip with tweezers and place it upright on paper to absorb excess water, add a small amount of anti-quenching agent (containing DAPI) to the slide, cover it with the coverslip, seal the slide, and store at 4℃ in the dark. Observe whether CsEVs can enter the host cells by laser confocal microscopy.

[0081] 2. CCK8 assay to detect cell proliferation activity

[0082] (1) Cell Culture

[0083] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 2 × 10⁻⁶. 4 Cells were seeded at a rate of 100 μL / well into four 96-well plates and incubated at 37°C in a 5% CO2 incubator. After cell attachment, the culture medium was aspirated.

[0084] (2) Co-cultivation

[0085] The RBE cells and HuCCT1 cells were each divided into 4 groups and incubated with 5 μg / mL CsEVs prepared in Example 1, 10 μg / mL CsEVs prepared in Example 1, and 20 μg / mL CsEVs prepared in Example 1, respectively. An equal volume of PBS was used as a control. Each group had 5 replicates and was cultured in a 37°C, 5% CO2 cell culture incubator for 0 h, 24 h, 48 h, and 72 h, respectively.

[0086] (3) Detection

[0087] The serum-free 1640 medium and CCK8 reagent were mixed at a volume ratio of 10:1 to prepare the test solution. The old medium was removed with a pipette, and the test solution was added at a rate of 100 μL / well. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 1 hour in the dark. The absorbance of the cells at a wavelength of 450 nm was measured using an ELISA reader, and the cell proliferation activity was compared by measuring the absorbance values.

[0088] 3. EdU-488 cell proliferation detection

[0089] (1) Cell Culture

[0090] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 Cells / mL were seeded into 6-well plates and cultured in a 37°C, 5% CO2 incubator.

[0091] (2) Co-cultivation

[0092] After the cells were cultured overnight and returned to normal, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0093] (3) Detection

[0094] RBE cells and HuCCT1 cells were stained using the EdU-488 cell proliferation assay kit, and changes in proliferating cells were observed under a fluorescence microscope.

[0095] 4. Plate colony formation assay for cell proliferation

[0096] (1) Cell Culture

[0097] RBE cells and HuCCT1 cells were seeded into 6-well plates, respectively.

[0098] (2) Co-cultivation

[0099] After the cells adhered, RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0100] (3) Detection

[0101] Cells were digested using trypsin and made into a cell suspension. They were then seeded into 6-well plates at a density of 1000 cells per well. The culture medium was changed every 3 days, and the cells were incubated statically for 1 to 2 weeks until visible clones appeared in the culture dish. The culture was then terminated.

[0102] Cells were fixed with 4% v / v paraformaldehyde (Beyotime, LOT: P0099) and stained with 1 mL of 1% crystal violet for 20 min. Finally, images were taken using a scanner, and the cell clone count was performed using ImageJ.

[0103] 5. Flow cytometry to detect cell cycle distribution

[0104] (1) Cell Culture

[0105] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 Cells / mL were seeded into 6-well plates and cultured in a cell culture incubator.

[0106] (2) Co-cultivation

[0107] After the cells were cultured overnight and returned to normal, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0108] (3) Detection

[0109] Adherent cells were digested with EDTA-free trypsin (to avoid false positives, the digestion time should not be too long), and 5 × 10⁶ cells were collected from each well. 5 Cells were centrifuged at 1500 rpm for 5 min in different flow cytometry tubes, and the supernatant was discarded. The cell pellet was washed twice with PBS. Using a cell cycle kit (manufacturer: Linko Biotech, catalog number: CCS012), 1 mL of buffer A was added to the cell pellet, and the cells were gently pipetted to form a cell suspension. Then, 10 μL of buffer B was added, mixed well, and incubated at room temperature in the dark for 30 min.

[0110] Cell cycle detection was performed using flow cytometry.

[0111] 6. Cell scratch assay to detect cell migration ability

[0112] (1) Preprocessing

[0113] Make markings on the back of the 6-well plate beforehand.

[0114] (2) Cell Culture

[0115] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 Cells / mL were seeded into 6-well plates and cultured in a cell culture incubator.

[0116] (3) Co-cultivation

[0117] After the cells adhered, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0118] (4) Detection

[0119] When the cells formed a monolayer, a 200 μL sterile pipette tip was used to draw a straight line down between the cells using a ruler. After adhering to the well, the cells were slowly washed twice with PBS, and then 2 mL of serum-free 1640 medium was added to each well. The width of the cell scratch was observed and photographed under a microscope at 0 h. The cells were then placed in an incubator, and the healing of the cell scratches was observed and recorded under a microscope every 24 h.

[0120] 7. Transwell assay to detect cell migration and invasion capabilities

[0121] (1) Preprocessing

[0122] Transwell chambers for detecting cell migration: Place the transwell chambers into a 24-well plate and hydrate the basement membrane with 100 μL of serum-free 1640 medium for 20 min.

[0123] Transwell chambers for detecting cell invasion: Matrigel (corning, LOT: 356234) was diluted with serum-free 1640 medium at a volume ratio of 1:8. 60 μL of the diluted Matrigel was added to the upper chamber, coating the upper surface of the transwell chamber filter membrane. The chamber was then incubated at 37°C with 5% CO2 for 5 hours. After the Matrigel solidified, 100 μL of serum-free 1640 medium was used to hydrate the basement membrane for 20 minutes.

[0124] (2) Cell Culture

[0125] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 The cells were inoculated at a density of 1 / mL into 6-well plates and incubated at 37°C in a 5% CO2 incubator.

[0126] (3) Co-cultivation

[0127] After the cells adhered, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0128] (4) Detection

[0129] Discard the old culture medium in the transwell chamber, and take the RBE cells and HuCCT1 cells obtained from the previous co-culture step, respectively, and adjust the concentration to 1×10⁻⁶ using FBS-free 1640 medium. 6100 μL of cell suspension was seeded into the upper chamber of the Transwell chamber, and 600 μL of 1640 medium containing 10% v / v FBS was added to the lower chamber. The chamber was then incubated at 37°C and 5% CO2 for 24 h.

[0130] Afterwards, the culture medium in the upper chamber was aspirated, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then soaked in 1% crystal violet staining solution for 15 minutes. After gently wiping the surface of the upper chamber with a cotton swab to remove any cells that had not penetrated the membrane, the cells were washed twice with PBS and then allowed to air dry.

[0131] Observation was performed under a microscope. Five fields of view were randomly selected from each membrane for observation and photography. The number of cells that had permeated the membrane was counted using ImageJ. Each experiment was repeated three times with three replicates.

[0132] 8. RT-qPCR target sequence

[0133] (1) Cell Culture

[0134] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 The cells were inoculated at a density of 1 / mL into 6-well plates and incubated at 37°C in a 5% CO2 incubator.

[0135] (2) Co-cultivation

[0136] After the cells adhered, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0137] (3) Total RNA extraction and reverse transcription

[0138] Total RNA was extracted from cells using an RNA extraction kit, and the purity and concentration of the total RNA were determined using a UV spectrophotometer. cDNA was synthesized using 1 μg of total RNA via reverse transcription.

[0139] (4) qPCR detection

[0140] Using the obtained cDNA as a template, qPCR amplification was performed using SYBR Green-I to detect the expression levels of cell cycle-related regulatory factors, with β-actin as an internal control. The primers used are shown in Table 1, and the qPCR reaction system is shown in Table 2.

[0141] Table 1 Primers used for qPCR detection

[0142]

[0143]

[0144] Table 2 qPCR reaction system

[0145]

[0146] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 30 s, and 40 cycles of amplification.

[0147] The experiment was conducted with 3 replicate wells, repeated 3 times, using 2 -ΔΔCt Calculations were performed using relative quantitative analysis.

[0148] 9. Western blot

[0149] (1) Cell Culture

[0150] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 The cells were inoculated at a density of 1 / mL into 6-well plates and incubated at 37°C in a 5% CO2 incubator.

[0151] (2) Co-cultivation

[0152] After the cells adhered, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 48 h, with an equal volume of PBS as the control group.

[0153] (3) Sample preparation

[0154] Total protein was extracted from control group and CsEVs-treated cells. Protein concentration was measured according to the BCA protein concentration kit instructions. All protein samples were adjusted to equal concentration, thoroughly mixed, and then loaded with loading buffer. The mixture was heated in boiling water for 10 min before loading.

[0155] (4) Detection

[0156] Electrophoresis was performed using a 10% SDS-PAGE gel, followed by membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and development. The expression of cell cycle-related proteins and epithelial-mesenchymal transition (EMT)-related proteins was detected. The primary antibodies used are as follows: anti-CDK2 (manufacturer: CST, catalog number: #18048), anti-CDK4 (manufacturer: CST, catalog number: #12790), anti-CDK6 (manufacturer: CST, catalog number: #3136), anti-CyclinD1 (manufacturer: CST, catalog number: #55506), anti-CyclinD3 (manufacturer: CST, catalog number: #2936), anti-E-cadherin (manufacturer: proteintech, catalog number: #60335), anti-N-cadherin (manufacturer: CST, catalog number: #13116), anti-vimentin (manufacturer: CST, catalog number: #5741), anti-slug (manufacturer: CST, catalog number: #9585), and anti-GAPDH (manufacturer: CST, catalog number: #5174).

[0157] 10. Data Analysis

[0158] Statistical analysis was performed using GraphPad Prism 8.0 software. The t-test was used to compare the means of the two samples. Data are expressed as mean ± standard deviation (Mean ± SD). Differences were considered statistically significant in the following cases: *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001.

[0159] II. Experimental Results

[0160] 1. Extracellular vesicles of Clonorchis sinensis can be endocytosed by bile duct cancer cells.

[0161] like Figure 3 As shown, red fluorescent signals were clearly visible around the nuclei (blue) of cholangiocarcinoma cells as the incubation time increased, indicating that CsEVs could be internalized by cholangiocarcinoma cells in a time-dependent manner.

[0162] 2. Extracellular vesicles of Clonorchis sinensis promote the proliferation of bile duct cancer cells.

[0163] like Figure 4 As shown in A, CsEVs can promote the proliferation of cholangiocarcinoma cell lines RBE and HuCCT1, with the optimal concentration being 10 μg / mL and the optimal treatment time being 48 h.

[0164] like Figure 4As shown in B and C, compared with the control group, CsEVs treatment increased the number of EdU-positive cells and colony formation, indicating that CsEVs promoted the proliferation of cholangiocarcinoma cells.

[0165] like Figure 4 As shown in D, treatment of cholangiocarcinoma cells with CsEVs increased the number of cholangiocarcinoma cells in both the S and G2 / M phases, and improved the proliferation index.

[0166] 3. Extracellular vesicles of Clonorchis sinensis promote the proliferation of bile duct cancer cells by regulating cell cycle-related molecules.

[0167] like Figure 5 As shown in A-D, in RBE cells and HuCCT1 cells treated with CsEVs, the levels of cyclins Cyclin D1 and Cyclin D3, as well as cyclin-dependent kinases CDK2, CDK4, and CDK6, were significantly increased, indicating that CsEVs stimulate the abnormal proliferation of cholangiocarcinoma cells by regulating cell cycle-related genes.

[0168] 4. Extracellular vesicles of Clonorchis sinensis promote the migration and invasion of cholangiocarcinoma cells.

[0169] like Figure 6 As shown in Figure A, compared with the PBS control group, CsEVs significantly improved the migration rate of RBE cells and HuCCT1 cells at 24 hours and 48 hours.

[0170] like Figure 6 As shown in B, after CsEVs treatment, the number of RBE cells and HuCCT1 cells that passed through the transwell chamber within 24 hours was significantly increased compared to the PBS negative control group.

[0171] like Figure 7 As shown, after CsEVs treatment, among the EMT-related marker molecules in RBE cells and HuCCT1 cells, the expression of epithelial marker E-cadherin was downregulated, while the expression of mesenchymal markers N-cadherin and Vimentin was upregulated; at the same time, the expression level of Slug protein, which regulates EMT marker molecules, was significantly upregulated.

[0172] These results indicate that CsEVs can promote the migration and invasion of cholangiocarcinoma cells, and Slug may play an important role in CsEVs-mediated EMT.

[0173] Example 4: Knocking down Slug inhibits extracellular vesicle-mediated EMT and cell migration and invasion in Clonorchis sinensis.

[0174] I. Experimental Methods

[0175] (1) Cell Culture

[0176] RBE cells and HuCCT1 cells were cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator, respectively. The cells were then digested with trypsin and suspended into a cell suspension, with the concentration adjusted to 5 × 10⁻⁶. 5 The cells were inoculated at a density of 1 / mL into 6-well plates and incubated at 37°C in a 5% CO2 incubator.

[0177] (2) Transfection

[0178] Based on the 1317bp to 1335bp (i.e., the nucleotide sequence 5'-CAAATCATTTCAACTGAAA-3') (SEQ ID NO.13) of the Slug gene (NCBI No.: 6591) as the target sequence, a Slug-specific siRNA (i.e., si-Slug) was designed, and the specific sequence is as follows:

[0179] si-Slug-Chain of Justice: 5'-CAAAUCAUUUCAACUGAAATT-3' (SEQ ID NO.14);

[0180] si-Slug-Ansense Chain: 5'-UUUCAGUUGAAAUGAUUUGTT-3' (SEQ ID NO.15).

[0181] A generic negative siRNA (i.e., si-Con) was used as a control for si-Slug, and its specific sequence is as follows:

[0182] si-Con-Chain of Justice: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.16);

[0183] si-Con-antisense chain: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.17).

[0184] *Regarding SEQ ID NO:14-17 in the “nucleotide or amino acid sequence listing” of this invention: According to the editing rules of WIPOSequence software, the nucleotide sequence must only contain the symbols listed in “Part 1 of Annex I of WIPO ST.26”. The base “t” is “u” in the RNA sequence. Therefore, the above SEQ ID NO:14-17 in this embodiment is substantially the same as SEQ ID NO:14-17 in the sequence listing.

[0185] After cell adhesion, the RBE cells and HuCCT1 cells were incubated with 10 μg / mL CsEVs prepared in Example 1 for 24 h. Then, si-Slug and si-Con were transfected into the CsEV-pretreated cells using Lipofectamine 3000. The final siRNA concentration was 100 nmol / L. 10 μL of siRNA was dissolved in 125 μL of Opti-MEM, gently mixed, and incubated for 5 min. 5 μL of Lipofectamine 3000 was dissolved in 125 μL of Opti-MEM, gently mixed, and incubated for 5 min. The siRNA and transfection reagents were then mixed, gently mixed, and incubated for 15 min. The transfection complex was added to 6-well plates, and the cells were cultured at 37°C in a 5% CO2 incubator for 48 h. Fresh medium was added 6 h after transfection.

[0186] (3) Western blot detection

[0187] Western blot analysis was performed according to the method described in Example 3.

[0188] (4) RT-qPCR detection

[0189] RT-qPCR detection was performed according to the method in Example 3, using the upstream primer Slug-F: 5'-CGAACTGGACACACATACAGTG-3' (SEQ ID NO.18) and the downstream primer Slug-R: 5'-CTGAGGATCTCTGGTTGTGGT-3' (SEQ ID NO.19) to detect Slug.

[0190] (5) Transwell chamber detection

[0191] Transwell chamber testing was performed according to the method described in Example 3.

[0192] II. Experimental Results

[0193] like Figure 8 As shown in A and B, compared with the si-Con group, the expression levels of Slug protein and mRNA decreased 48 h after si-Slug transfection, the expression level of E-cadherin was restored, while the expression levels of N-cadherin and Vimentin were downregulated. Figure 8 As shown in Figure C, knocking down Slug weakened the promoting effect of CsEVs on the migration and invasion of cholangiocarcinoma cells. Compared with the si-Con group, the number of RBE cells and HuCCT1 cells that crossed the transwell chamber within 24 hours after si-Slug transfection for 48 hours was reduced.

[0194] These results indicate that knocking down Slug can inhibit CsEVs-mediated EMT and cell migration and invasion. CsEVs can induce EMT in cholangiocarcinoma cells, thereby enabling tumors to migrate and invade, and Slug plays a key role in CsEVs-mediated EMT and cholangiocarcinoma metastasis.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of an agent that inhibits the expression of a Slug gene and / or a Slug protein in cholangiocarcinoma cells in the manufacture of a therapeutic drug for clonorchis-induced cholangiocarcinoma, characterized in that, The cholangiocarcinoma is a cholangiocarcinoma induced by Clonorchis sinensis extracellular vesicles, and the agent comprises an siRNA with a Slug gene as a target, wherein a nucleotide sequence of a sense strand of the siRNA is as shown in SEQ ID NO. 14, and a nucleotide sequence of an antisense strand is as shown in SEQ ID NO.

15.

2. A medicament for treating clonorchis sinensis-induced cholangiocarcinoma, characterized by, The drug comprises an siRNA with a Slug gene as a target, wherein a nucleotide sequence of a sense strand of the siRNA is as shown in SEQ ID NO. 14, and a nucleotide sequence of an antisense strand is as shown in SEQ ID NO. 15.

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