Use of lepr gene or protein as a target in the manufacture of a therapeutic product for cholangiocellular carcinoma

Abstract

The present application relates to the field of biological medicine, in particular to the use of LEPR gene or protein as a target in the preparation of cholangiocellular carcinoma treatment product, the present application finds that LEPR can be used as a therapeutic target and a prognostic marker of cholangiocellular carcinoma, research shows that the up-regulation of LEPR expression level inhibits the proliferation, migration and invasion of CCA cells, which indicates that LEPR can be used as a target of cholangiocellular carcinoma treatment drug, and is used for preparing or screening cholangiocellular carcinoma treatment drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the use of LEPR gene or protein as a target in the preparation of cholangiocellular carcinoma treatment products. BACKGROUND

[0002] Cholangiocarcinoma (CCA) is a malignant tumor occurring in the biliary tree and derived from cholangiocytes. Global cancer statistics show that the incidence of CCA is on the rise. Surgery is the only curative treatment for CCA, but it is only suitable for early-stage patients in good physical condition. Most patients are found to be in the advanced stage of the disease, and can only receive radiotherapy / chemotherapy or immunotherapy. Since the disease is often discovered in the late stage, the prognosis of patients is poor, and the 5-year survival rate is less than 20%. Therefore, exploring new therapeutic targets and finding prognostic markers are of great significance to improve the survival of CCA patients.

[0003] Obesity has become an important risk factor for various cancers, including CCA. Epidemiological studies have shown that obesity promotes carcinogenesis through chronic inflammation, metabolic disorders, and changes in signaling pathways. Leptin receptor (LEPR) is a transmembrane protein mainly expressed in the hypothalamus and peripheral tissues, and plays a key role in regulating energy homeostasis, appetite, and body weight. Dysregulation of the leptin LEPR axis is associated with the pathogenesis of obesity, metabolic disorders, and inflammatory diseases. The leptin LEPR axis can promote tumor proliferation, angiogenesis, and immune escape by regulating multiple signaling pathways. However, the role of LEPR in CCA has not been fully studied. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide the use of LEPR gene or protein as a target in the preparation of cholangiocellular carcinoma treatment products, to solve the problems in the prior art.

[0005] To achieve the above-mentioned purposes and other related purposes, the present application first provides the use of LEPR gene or protein as a target in the preparation or screening of cholangiocellular carcinoma treatment products or prognostic evaluation products.

[0006] The present application also provides the use of LEPR gene, LEPR protein, or LEPR gene overexpression reagent in the preparation of cholangiocellular carcinoma treatment products.

[0007] The present application also provides the use of LEPR gene, LEPR protein, or LEPR overexpression reagent in the preparation of products with any of the following effects:

[0008] 1) inhibiting the proliferation of cholangiocarcinoma cells;

[0009] 2) inhibiting the colony formation of cholangiocarcinoma cells;

[0010] 3) inhibiting the invasion or migration of cholangiocarcinoma cells;

[0011] 4) inducing cell cycle arrest.

[0012] The present application also provides the use of a substance specifically binding to the LEPR protein or the encoding gene thereof in the preparation of a cholangiocellular carcinoma prognosis evaluation product.

[0013] The present application also provides a composition for treating cholangiocarcinoma, the effective substance of which contains the LEPR gene, LEPR protein or LEPR overexpression reagent and a pharmaceutically acceptable carrier or excipient.

[0014] As described above, the use of the LEPR gene or protein of the present application as a target in the preparation of a cholangiocellular carcinoma treatment product has the following beneficial effects: LEPR has been found to be a therapeutic target and a prognosis marker of cholangiocarcinoma, and the present application research shows that the up-regulation of LEPR expression level inhibits the proliferation, migration and invasion of CCA cells, indicating that LEPR can be used as a target of CCA therapeutic drugs for the preparation or screening of CCA therapeutic drugs. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Expression difference of LEPR protein in CCA and adjacent non-tumor (ANT) tissues in the researcher cohort. (A) Representative images of expression difference of LEPR in CCA and ANT tissues (magnification 200x); (B) Expression difference of LEPR in 60 cases of CCA and 27 cases of ANT tissues; (C) Expression difference of LEPR in 27 pairs of matched CCA and ANT tissues; (D) Western blot detection of protein expression of LEPR in 5 pairs of matched fresh CCA and ANT surgical specimens. n = 10.

[0016] Figure 2 Exploration of the relationship between LEPR expression level and clinical pathological characteristics of CCA patients based on the researcher cohort. (A-E) Correlation of LEPR expression level with patient age, gender, BMI, T stage and pathological stage. In D figure, the higher the T stage, the higher the malignancy degree, and the lower the LEPR expression. In E figure, Stage I, II, III and IV are pathological stages, the higher the stage, the higher the malignancy degree, and the lower the LEPR expression.

[0017] Figure 3 Relationship between high and low expression of LEPR and prognosis of CCA patients in the researcher cohort. (A) Relationship between high and low expression of LEPR and survival of CCA patients in the researcher cohort; (B) Forest plot based on single factor and multiple factor COX regression analysis of the researcher cohort data.

[0018] Figure 4 Construction of CCA cell model stably up-regulating LEPR. (A) Western blot detection of LEPR expression in 5 CCA cells; (B) qRT-PCR detection of LEPR protein expression in different groups of SK-CHA-1 cells; (C) Western blot detection of LEPR protein expression in different groups of SK-CHA-1 cells; (D) qRT-PCR detection of LEPR protein expression in different groups of KMBC cells; (E) Western blot detection of LEPR protein expression in different groups of KMBC cells. n = 3.

[0019] Figure 5 Effect of up-regulating LEPR on CCA cell proliferation. (A) CCK-8 method for detecting the effect of LEPR overexpression on SK-CHA-1 cell proliferation; (B) CCK-8 method for detecting the effect of LEPR overexpression on KMBC cell proliferation; (C) Plate colony formation experiment to study the effect of LEPR overexpression on the proliferation ability of SK-CHA-1 cells; (D) Plate colony formation experiment to study the effect of LEPR overexpression on the proliferation ability of KMBC cells. n = 3.

[0020] Figure 6 Effect of up-regulating LEPR on CCA cell cycle. (A) Flow cytometry detection of the effect of LEPR overexpression on the cell cycle of SK-CHA-1 cells; (B) Flow cytometry detection of the effect of LEPR overexpression on the cell cycle of KMBC cells. n = 3.

[0021] Figure 7 Effect of up-regulating LEPR on SK-CHA-1 and KMBC cell migration and invasion. (A) Scratch test to detect the effect of LEPR overexpression on the migration ability of SK-CHA-1 cells; (B) Scratch test to detect the effect of LEPR overexpression on the migration ability of KMBC cells. n = 3. (C) Detection of the effect of LEPR overexpression on the migration and invasion ability of SK-CHA-1 cells; (D) Detection of the effect of LEPR overexpression on the migration and invasion ability of KMBC cells. n = 3.

[0022] In the drawings:

[0023] *P <0.05, **P <0.01, ***P <0.001; eCCA: extrahepatic cholangiocarcinoma tissue, ANT: adjacent non-tumor tissue; LV-Control: negative control; LV-LEPR: LEPR lentivirus overexpression plasmid. DETAILED DESCRIPTION

[0024] The present application explores the potential role of LEPR in CCA, aiming to provide a new potential therapeutic target for cholangiocarcinoma.

[0025] The present application verifies the expression of LEPR and its correlation with the clinicopathological features of patients and the survival of patients from the researcher cohort. Subsequently, the role of LEPR gene in the development of CCA is confirmed from the perspective of cell function. By constructing the LEPR overexpression stable cell strain, and through cell proliferation and colony formation experiment, cell cycle, cell scratch, cell migration experiment and cell invasion experiment, the results show that the up-regulation of LEPR expression level can inhibit the proliferation, migration and invasion of CCA cells. It is shown that LEPR can be used as a target for CCA therapeutic drugs.

[0026] The present application first provides the use of LEPR (leptin receptor) gene or protein as a target in the preparation or screening of cholangiocarcinoma treatment products or prognosis evaluation products.

[0027] In some embodiments of the present application, the cholangiocarcinoma is extrahepatic cholangiocarcinoma.

[0028] The present application also provides the use of LEPR gene, LEPR protein or LEPR gene overexpression reagent in the preparation of cholangiocarcinoma treatment products.

[0029] In some embodiments of the present application, the LEPR gene is a LEPR gene fragment or a LEPR full-length sequence.

[0030] In some embodiments of the present application, the LEPR protein is a LEPR complete protein or a protein fragment; the LEPR protein is a natural LEPR protein or a recombinant LEPR protein.

[0031] The LEPR gene and LEPR protein can be directly administered to directly increase the level of LEPR gene or protein in cells to achieve a therapeutic effect.

[0032] In some embodiments of the present application, the LEPR overexpression reagent is selected from a nucleic acid construct containing a LEPR gene or a virus with a LEPR gene integrated in the genome.

[0033] In some embodiments of the present application, the nucleic acid construct containing a LEPR gene can be obtained by cloning a LEPR gene fragment (such as the sequence shown in SEQ ID NO. 1) into a known vector.

[0034] In some embodiments of the present application, the LEPR gene nucleic acid construct is a mammalian expression vector.

[0035] Further, the mammalian expression vector is selected from one or more of a LEPR gene adeno-associated viral vector, a LEPR gene lentiviral vector, a LEPR gene adenoviral vector, or a LEPR gene retroviral vector.

[0036] In an embodiment of the present application, a lentiviral vector containing a LEPR gene is specifically constructed. The nucleotide sequence of the LEPR gene is shown in SEQ ID NO. 1. The lentiviral vector of the LEPR gene disclosed in the present application is obtained by cloning a LEPR gene fragment into a known vector, which is a lentiviral vector. After the lentiviral vector of the LEPR gene is packaged into an infectious viral particle, it infects cells and further overexpresses the LEPR gene.

[0037] In some embodiments of the present application, the virus integrating the LEPR gene into the genome is selected from one or more of a lentivirus, an adenovirus, an adeno-associated virus, or a retrovirus.

[0038] The cholangiocarcinoma treatment drug is a molecule that can specifically promote the transcription or translation of the LEPR gene, or can specifically promote the expression of the LEPR gene or the activity of the LEPR protein, thereby increasing the content level of the LEPR protein in the patient's body.

[0039] The cholangiocarcinoma treatment drug prepared by the LEPR gene, the LEPR protein, or the LEPR gene overexpression reagent includes but is not limited to: a nucleic acid molecule, an antibody drug, a polypeptide, a protein, an adeno-associated virus, or a lentivirus.

[0040] The administration amount of the cholangiocarcinoma treatment drug is a dose sufficient to up-regulate the transcription or translation of the LEPR gene, or a dose sufficient to up-regulate the expression or activity of the LEPR protein. So that the expression of the LEPR gene is up-regulated by at least 50%, 80%, 90%, 95%, or 99%.

[0041] The LEPR overexpression reagent refers to a substance that has an up-regulation effect on LEPR. The up-regulation effect on LEPR includes but is not limited to:

[0042] 1) promoting the expression of the LEPR gene or the activity of the LEPR protein;

[0043] 2) using overexpression to increase the expression level of the LEPR gene.

[0044] Promoting the activity of the LEPR protein refers to increasing the activity of the LEPR protein. Preferably, the activity of the LEPR protein is increased by at least 10% compared to before up-regulation, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0045] Promoting the expression of the LEPR gene can be specifically promoting the transcription or translation of the LEPR gene, and can be specifically referring to increasing the transcription or expression of the LEPR gene by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by 70%, and most preferably by at least 90%.

[0046] The present application also provides the use of the LEPR gene, the LEPR protein or the LEPR overexpression reagent in the preparation of a product having any of the following effects:

[0047] 1) inhibiting the proliferation of cholangiocarcinoma cells;

[0048] 2) inhibiting the colony formation of cholangiocarcinoma cells;

[0049] 3) inhibiting the invasion or migration of cholangiocarcinoma cells;

[0050] 4) inducing cell cycle arrest.

[0051] In some embodiments of the present application, the expression level of LEPR can be increased by transfecting an overexpression plasmid.

[0052] In some embodiments of the present application, the expression level of LEPR can be increased by constructing a lentiviral vector.

[0053] The cholangiocarcinoma treatment drug necessarily includes the LEPR gene, the LEPR protein or the LEPR overexpression reagent, and the LEPR gene, the LEPR protein or the LEPR overexpression reagent are used as the effective components for the above-mentioned effects.

[0054] In the cholangiocarcinoma treatment drug, the effective components for the above-mentioned effects can only be the LEPR gene, the LEPR protein or the LEPR overexpression reagent, or can also include other molecules that can have similar effects.

[0055] The LEPR gene, the LEPR protein or the LEPR overexpression reagent are the only effective components or one of the effective components of the cholangiocarcinoma treatment drug.

[0056] The cholangiocarcinoma treatment drug can be a single-component substance or a multi-component substance.

[0057] The product is mainly aimed at mammals. The mammals are preferably rodents, even-toed ungulates, odd-toed ungulates, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0058] The product includes but is not limited to drugs, health products, food, etc.

[0059] The present application also provides the use of a substance specifically binding to the LEPR protein or the encoding gene thereof in the preparation of a cholangiocarcinoma prognosis evaluation product.

[0060] The TNM staging of extrahepatic bile duct cancer is composed of T staging, N staging and M staging, and each staging is divided into different degrees, for example, T1, T2, T3, T4 in T staging, N0, N1, N2 in N staging, M0 and M1 in M staging, and the greater the value, the more serious the disease.

[0061] In some embodiments of the present application, the prognosis evaluation product is used to detect the content level of LEPR gene or protein.

[0062] The present application also provides a method for treating bile duct cancer, which comprises administering LEPR gene, LEPR protein or LEPR overexpression reagent to a subject.

[0063] The subject can be a mammal. The mammal is preferably a rodent, an even-toed ungulate, an odd-toed ungulate, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human.

[0064] The subject can be a patient suffering from bile duct cancer or an individual expecting to prevent or alleviate bile duct cancer.

[0065] The LEPR gene, LEPR protein or LEPR overexpression reagent can be administered to the subject before, during or after the treatment of bile duct cancer.

[0066] The present application also provides a composition for treating bile duct cancer, wherein the effective substance contains LEPR gene, LEPR protein or LEPR overexpression reagent and a pharmaceutically acceptable carrier or excipient.

[0067] The expression amount of LEPR protein in the patient's body can be increased by directly administering LEPR protein or administering a product promoting LEPR expression (e.g. overexpression reagent), or both.

[0068] The drug for treating bile duct cancer must contain LEPR gene, LEPR protein or LEPR overexpression reagent, and LEPR gene, LEPR protein or LEPR overexpression reagent is used as the effective component for the aforementioned function.

[0069] "Pharmaceutically acceptable" means that when the molecular entity and the composition are properly administered to animals or humans, they do not produce adverse, allergic or other untoward reactions.

[0070] A "pharmaceutically acceptable carrier or adjuvant" shall mean a non-toxic substance that is compatible with the effective ingredient and does not substantially interfere with the effectiveness of the pharmaceutical. The pharmaceutically acceptable adjuvant includes one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption accelerators, surfactants, adsorptive carriers or lubricants. Specific examples of some substances that can be used as the pharmaceutically acceptable carrier or adjuvant are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffered solutions, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0071] The present application now will be described by way of specific examples, which are intended to illustrate but not limit the application. Other advantages and benefits of the present application will become apparent to those skilled in the art upon reading the foregoing specification and inspecting the accompanying drawings.

[0072] Before further description of the application, it is to be understood that the application is not limited to the particular specific embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as to the scope of the present application. In this specification and in the claims that follow, reference will be made to a number of terms which shall have the meanings defined below.

[0073] When numerical ranges are given, it should be understood that every numerical range encompasses any number falling within the range, including the endpoints, and any intervening range governed by the same limits. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described herein.

[0074] The biological role of LEPR in CCA was studied by the following examples, and the results showed that LEPR expression up-regulation inhibited the proliferation, migration and invasion of CCA cells. The inventors verified the expression of LEPR and its correlation with the clinical pathological characteristics and survival of patients in the inventors' cohort. Subsequently, the inventors confirmed the role of LEPR gene in the occurrence and development of CCA from the perspective of cell function. By constructing the lentivirus of the target gene, the lentivirus was transfected into CCA cells, and the expression of the target gene at the mRNA and protein levels in the two groups of CCA cell lines was detected. Subsequently, cell proliferation, migration, invasion and cell cycle were detected by cell function experiments, and the results showed that the proliferation degree of CCA cells in the LEPR OE group was significantly higher than that in the control group. Based on the above research results, further exploration and development of new methods for diagnosis and treatment of the gene can provide more choices for the diagnosis and treatment of CCA patients.

[0075] Example 1 Difference in expression of LEPR protein in CCA and ANT in the inventors' cohort

[0076] This example was based on 60 cases of CCA patients' tissues collected retrospectively in the inventors' cohort, and IHC detection found that LEPR was significantly lower expressed in CCA tissues than in control ANT tissues (P<0.001, Figure 1 A-C) Western blot was used to detect the protein expression of LEPR in 5 pairs of fresh CCA and ANT surgical specimens, and it was also confirmed that LEPR was significantly lower expressed in CCA tissues than in control ANT tissues (P<0.001, Figure 1 D).

[0077] Example 2 Relationship between LEPR expression and clinical pathological characteristics of CCA patients based on the inventors' cohort

[0078] To clarify the clinical value of LEPR in Chinese CCA patients, the inventors analyzed the correlation between the expression level of LEPR and the clinical pathological characteristics of CCA patients based on the IHC results of 60 patients in the inventors' cohort, and the results showed that the lower the expression of LEPR, the higher the T stage and pathological stage (A-E), which indicated that the lower the expression of LEPR, the more serious the condition of CCA patients. Figure 2 A-E), which indicated that the lower the expression of LEPR, the more serious the condition of CCA patients.

[0079] Example 3 Relationship between LEPR expression and prognosis of CCA patients

[0080] The inventors analyzed the correlation between the expression level of LEPR and the survival of CCA patients based on the IHC results of 60 patients in the inventors' cohort. The Kaplan-Meier survival analysis results showed that compared with the high expression group of LEPR, the CCA patients with low expression of LEPR had shorter overall survival (OS) (Figure 3 A) Single factor COX regression analysis showed that T stage, pathological stage and LEPR expression level were the prognostic factors of CCA patients, and multi-factor COX further confirmed that LEPR expression and T stage were the independent prognostic factors of CCA patients (all P<0.05, Figure 3 B).

[0081] Example 4 Construction of CCA cell model stably up-regulating LEPR

[0082] To further study the role of LEPR in CCA, the expression level of LEPR in five CCA cell lines was detected by Western blot, including three eCCA cell lines (QBC939, SK-CHA-1 and KMBC) and two iCCA cell lines (RBE and HuCCT1). The results showed that the expression of LEPR in eCCA cell lines SK-CHA-1 and KMBC was lower than that in iCCA cell lines RBE and HuCCT1. Figure 4 A) Therefore, SK-CHA-1 and KMBC cells were selected as tool cells for subsequent experiments, and SK-CHA-1 cell models and KMBC cell models stably overexpressing LEPR were constructed, and were confirmed by qRT-PCR and Western blot.

[0083] (1) Construction of overexpression plasmid vector

[0084] To investigate the effect of LEPR on CCA cells, overexpression targeting LEPR was used to overexpress LEPR in KMBC and SK-CHA-1 cell lines. STR identification was performed before the above CCA cell experiments, and there was no mycoplasma contamination. The specific construction method is as follows: LEPR overexpression plasmid was constructed using GV492 vector, and the element sequence was: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin. After digestion with BamHI / AgeI, the target gene sequence (such as SEQ ID NO. 1) was inserted. The above were synthesized by Shanghai Jikai Biological Technology Co., Ltd., and LEPR overexpression plasmid (LV-LEPR) and control (LV-Control) plasmid were obtained.

[0085]

[0086]

[0087]

[0088] (2) Lentivirus packaging

[0089] The lentivirus packaging was completed by Shanghai Jikai Biotechnology Co., Ltd., and the specific packaging process was carried out according to the instructions of Shanghai Jikai Biotechnology Co., Ltd. The general process is as follows: the tool vector plasmid carrying the target gene or target sequence, virus packaging auxiliary plasmid Helper1.0 and virus packaging auxiliary plasmid Helper2.0 are co-transfected into 293T cells, and the virus is harvested (i.e. unpurified cell supernatant) 48-72h after transfection. Concentration and purification of high-titer lentivirus stock solution, determination of lentivirus indicators meet the quality standards.

[0090] (3) Lentivirus infection

[0091] The well-grown KMBC and SK-CHA-1 cell lines were digested and centrifuged and inoculated in a six-well plate. When the cell confluence reached 30% and entered the growth logarithmic phase, the transfection experiment was started. According to the instructions attached by Jikai, the required virus volume was determined according to the multiplicity of infection (MOI) (add virus volume per well = MOI * cell number / virus titer).

[0092] (4) Transfection efficiency evaluation

[0093] The transfection effect was evaluated by observing the expression of GFP in cells 72h after transfection, RT-qPCR and Western Blot results.

[0094] The results are shown in Figure 4 , which show that the expression of LEPR in eCCA cell lines SK-CHA-1 and KMBC is low Figure 4 (A). Therefore, SK-CHA-1 and KMBC cells were selected as tool cells for subsequent experiments, and SK-CHA-1 cell models stably overexpressing LEPR Figure 4 (B-C) and KMBC Figure 4 (D-E) cell models were constructed, and were confirmed by qRT-PCR and Western blot.

[0095] Example 5 Effect of LEPR on the malignant behavior of CCA cell lines

[0096] 1. CCK-8 experiment, method as follows: take the state of the best cells after trypsin digestion, adjust the cell suspension concentration to 2.5x10 3cells / 100 μl, and inoculated in 96-well plates (100 μl per well). Incubate in a 37°C, 5% CO2 incubator for 24 hours to allow complete adhesion. At 1 / 2 / 3 days after intervention, remove the culture medium in the wells, and add 10 μl of CCK-8 working solution (diluted 1:10 with the basic culture medium) to each well. Incubate at 37°C for 120 minutes in the dark, and observe the color development process every 30 minutes. Use a M5 microplate reader preheated to 37°C, set the dual-wavelength detection mode (450 nm detection wavelength / 630 nm reference wavelength), and measure the absorbance value of each well. Convert the OD value of the experimental group to the relative proliferation rate (%) = (experimental group OD / control group OD) x 100%.

[0097] The CCK-8 experiment results show that, compared with the LV-Control group, the SK-CHA-1 cells in the LV-LEPR group have no significant difference in proliferation ability at 1 day, but the SK-CHA-1 cells in the LV-LEPR group significantly decrease in proliferation at 2 days and 3 days (P < 0.001) Figure 5 A). In KMBC cells, the proliferation in the LV-LEPR group is significantly inhibited at different detection points (P < 0.001) Figure 5 B).

[0098] 2. Plate colony formation experiment, method as follows: (1) Prepare a single cell suspension and adjust the density to 3 x 10 2 cells / ml, and inoculate 2 ml per well in a 6-well plate. Maintain a 37°C constant temperature environment for 10-14 days, and replace the complete culture medium containing 10% FBS every 72 hours. When the diameter of the visible clone mass is > 0.5 mm, discard the culture medium, and gently rinse three times with pre-cooled PBS at 4°C. Add 4% paraformaldehyde for fixation for 15 minutes, and 0.1% crystal violet (prepared with methanol) for staining for 20 minutes. Rinse with double-distilled water until the background is transparent, dry at room temperature, and then use an inverted microscope (4x objective) to collect images. Use Image J software to analyze the colony formation rate: number of colonies / inoculated cell number x 100% (threshold value set to > 50 cells / colony).

[0099] The colony formation experiment results show that the number of colonies formed by SK-CHA-1 and KMBC cells in the LV-LEPR group is significantly lower than that in the LV-Control group (P < 0.001) Figure 5 C-D).

[0100] 3. Flow cytometry detection of cell staging experiment, method as follows: Digest and count the required cells, collect 5 x 10 5The cells to be detected were placed in a centrifuge tube, and the centrifuge tube was balanced and centrifuged. After centrifugation for 4 min, the centrifuge tube was taken out, the supernatant outside the cell precipitate was poured off, an appropriate amount of PBS was added using a pipette, and then the centrifuge tube was balanced and centrifuged again. After centrifugation, the supernatant was poured off. 1 mL of DNA staining solution was added to the centrifuge tube, and the centrifuge tube was mixed by blowing with a pipette. Then 10 μL of Permeabilization solution was taken out using a small-volume pipette, and was mixed by oscillation. The centrifuge tube was placed at room temperature for standing. After standing for 30 min, the centrifuge tube was taken out, and the appropriate flow cytometry program was adjusted for on-machine detection.

[0101] The flow cytometry results are as follows: cell cycle analysis shows that LEPR overexpression significantly reduces the proportion of SK-CHA-1 cells in the G2 / M phase (P<0.05) Figure 6 A), while in KMBC cells, LEPR overexpression significantly increases the proportion of cells in the S phase (P<0.05) Figure 6 B). These results show that LEPR upregulation can effectively inhibit the proliferation ability of CCA cells and induce cell cycle arrest.

[0102] 4. Cell scratch experiment, the method is as follows: 6-well plates are seeded at a density of 5×10 5 cells / well, and when the confluence reaches 90%, a 200-μl sterile needle is used to make a scratch perpendicular to the surface of the plate. The detached cells are removed by gently washing with PBS three times, and the medium containing 2% FBS is replaced. At 0 / 12 / 24 hours, the scratch width is recorded. The healing rate is calculated using the TScratch algorithm: (initial area-final area) / initial area×100%. The scratch experiment results show that after 24 hours, the scratch healing rate of SK-CHA-1 and KMBC cells in the LV-LEPR group is significantly lower than that in the NC group, and the inhibition of horizontal migration of SK-CHA-1 cells is more significant (P<0.001) Figure 7 A), while the migration inhibition of KMBC cells is smaller (P<0.05) Figure 7 B).

[0103] 5. Migration experiment, the method is as follows: 600 μl of complete medium is added to each lower chamber of a Transwell plate, and a sterile chamber is placed on the lower chamber, and there should be no air bubbles between the chamber and the lower chamber. The required cells are digested and counted (with basal medium), at 1.5×10 4Cells were seeded at a density suitable for the upper layer of the chambers and cultured in a cell culture incubator for 24 hours. The Transwell plate was removed, the chambers were taken out, and the cells were fixed with 4% paraformaldehyde for 30 minutes. After rinsing in PBS to remove paraformaldehyde, the cells were incubated in crystal violet in the dark for 30 minutes. The upper edge of the chamber was held by tweezers, and the cells were repeatedly rinsed in PBS to remove crystal violet and then air-dried. The chambers were photographed and counted in five fields of view under a microscope. The results showed that the number of migrating cells in the LV-LEPR group was significantly lower than that in the LV-Control group (P<0.01), indicating that LEPR upregulation inhibited vertical cell migration. Figure 7 CD).

[0104] 6. Invasion test, the method is as follows: Apply Matrigel matrix gel... Serum-free medium was diluted 1:8 and pre-coated into the upper chamber of Transwell chambers (8 μm pore size) (50 μl / well). Polymerization was carried out at 37°C for 2 hours to form a basement membrane model. A 1.5 × 10⁻⁶ m² / well was loaded into the upper chamber. 4 Cells / 150 μl serum-free suspension were injected into the lower chamber with 600 μl of chemotactic medium containing 20% ​​FBS. After 24 hours of culture, cells were fixed with 4% paraformaldehyde for 30 minutes, and the number of cells that had penetrated the membrane was quantitatively analyzed by staining with 0.5% crystal violet. The experimental results showed that, similar to the migration assay, the upregulation of LEPR further confirmed the inhibitory effect of LEPR on cell invasion ability. Figure 7 CD).

[0105] The results above show that upregulation of LEPR expression levels can inhibit the proliferation, migration, and invasion of CCA cells. This indicates that LEPR can serve as a therapeutic target in the preparation or screening of drugs for treating CCA, and in the preparation or screening of drugs that inhibit CCA proliferation, migration, and / or invasion.

[0106] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of the LEPR gene overexpression reagent in the preparation of cholangiocarcinoma treatment products, wherein the LEPR overexpression reagent is selected from nucleic acid constructs containing the LEPR gene or viruses whose genomes integrate the LEPR gene; the nucleotide sequence of the LEPR gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that, The cholangiocarcinoma mentioned is extrahepatic cholangiocarcinoma.

3. The use according to claim 1, characterized in that, The LEPR gene nucleic acid construct is a mammalian expression vector.

4. The use according to claim 3, characterized in that, The mammalian expression vector is selected from one or more of the following: LEPR gene adeno-associated virus vector, LEPR gene lentiviral vector, LEPR gene adenovirus vector, or LEPR gene retroviral vector.

5. The use according to claim 1, characterized in that, The virus whose genome integrates the LEPR gene is selected from one or more of lentiviruses, adenoviruses, adeno-associated viruses, or retroviruses.

6. The use of the LEPR overexpression reagent according to any one of claims 1 to 5 in the preparation of a product having any of the following effects: 1) Inhibits the proliferation of bile duct cancer cells; 2) Inhibits the clonal formation of bile duct cancer cells; 3) Inhibits the invasion or migration of bile duct cancer cells; 4) Induces cell cycle arrest.

7. Use of substances that specifically bind to the LEPR protein or its encoding gene in the preparation of prognostic assessment products for cholangiocarcinoma.

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