Application of LEPR gene or protein as target in preparation of cholangiocellular carcinoma treatment product

Abstract

The invention relates to the field of biological medicine, in particular to application of LEPR gene or protein serving as a target in preparation of cholangiocarcinoma treatment products, the fact that LEPR can serve as a cholangiocarcinoma treatment target and a prognostic marker at the same time is found, and research shows that the expression level of LEPR is up-regulated to inhibit proliferation, migration and invasion of CCA cells, so that the cholangiocarcinoma treatment effect is improved, and the cholangiocarcinoma treatment effect is improved. Therefore, the LEPR can be used as an action target of the cholangiocarcinoma treatment medicine and is used for preparing or screening the cholangiocarcinoma treatment medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of LEPR gene or protein as a target in the preparation of cholangiocarcinoma treatment products. Background Art

[0002] Cholangiocarcinoma (CCA) is a malignant tumor that arises from bile duct epithelial cells in the biliary tree. Global cancer statistics indicate an increasing incidence of CCA. Surgery is the only curative treatment for CCA, but it is only suitable for patients diagnosed in the early stages and in good physical condition. Most patients are diagnosed with the disease in the advanced stages, and are only eligible for chemotherapy, radiotherapy, or immunotherapy. Because the disease is often discovered in the late stages, the prognosis is poor, with a 5-year survival rate of less than 20%. Therefore, exploring new therapeutic targets and identifying prognostic markers are crucial for improving the survival of CCA patients.

[0003] Obesity has emerged as a significant risk factor for various cancers, including CCA. Epidemiological studies have shown that obesity promotes carcinogenesis through chronic inflammation, metabolic disorders, and altered signaling pathways. The leptin receptor (LEPR) is a transmembrane protein primarily expressed in the hypothalamus and peripheral tissues that 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 evasion by regulating multiple signaling pathways. However, the role of LEPR in CCA remains understudied. Summary of the Invention

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

[0005] To achieve the above objectives and other related objectives, the present invention first provides the use of LEPR gene or protein as a target in the preparation or screening of cholangiocarcinoma treatment products or prognosis assessment products.

[0006] The present invention also provides use of the LEPR gene, LEPR protein or LEPR gene overexpression reagent in preparing a product for treating cholangiocarcinoma.

[0007] The present invention also provides the use of a LEPR gene, a LEPR protein or a LEPR overexpression reagent in preparing a product having any of the following effects:

[0008] 1) Inhibit the proliferation of bile duct cancer cells;

[0009] 2) Inhibit the clone formation of bile duct cancer cells;

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

[0011] 4) Induce cell cycle arrest.

[0012] The present invention also provides use of a substance that specifically binds to LEPR protein or its encoding gene in preparing a product for evaluating the prognosis of cholangiocarcinoma.

[0013] The present invention also provides a composition for treating bile duct cancer, wherein the effective substance contains LEPR gene, LEPR protein or LEPR overexpression agent and pharmaceutically acceptable carrier or excipient.

[0014] As described above, the use of the LEPR gene or protein of the present invention as a target in the preparation of cholangiocarcinoma treatment products has the following beneficial effects: it was found that LEPR can be used as both a therapeutic target and a prognostic marker for cholangiocarcinoma. The research of the present invention showed that upregulation of LEPR expression levels inhibits the proliferation, migration and invasion of CCA cells, indicating that LEPR can be used as a target for CCA therapeutic drugs for the preparation or screening of CCA therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Differential expression of LEPR protein between CCA and adjacent non-tumor (ANT) tissues in the study cohort. (A) Representative images (200x magnification) showing differential expression of LEPR between CCA and ANT tissues; (B) Differential expression of LEPR between 60 CCA and 27 ANT tissues; (C) Differential expression of LEPR between 27 paired CCA and ANT tissues; (D) Western blot analysis of LEPR protein expression in five paired fresh CCA and ANT surgical specimens. n = 10.

[0016] Figure 2 We investigated the relationship between LEPR expression and clinicopathological characteristics in a cohort of CCA patients. (A, E) Correlation between LEPR expression and patient age, sex, BMI, T stage, and pathological stage. Figure D shows T stage; higher stage indicates increased malignancy and lower LEPR expression. Figure E shows Stages I, II, III, and IV, pathological stages; higher stage indicates increased malignancy and lower LEPR expression.

[0017] Figure 3 Relationship between high and low LEPR expression and prognosis of CCA patients in the study cohort. (A) Relationship between high and low LEPR expression and survival of CCA patients in the study cohort; (B) Forest plot of univariate and multivariate Cox regression analysis based on the study cohort data.

[0018] Figure 4 Construction of a CCA cell model with stably upregulated LEPR. (A) Western blot analysis of LEPR expression in five CCA cell lines; (B) qRT-PCR analysis of LEPR protein expression in SK-CHA-1 cells from different groups; (C) Western blot analysis of LEPR protein expression in SK-CHA-1 cells from different groups; (D) qRT-PCR analysis of LEPR protein expression in KMBC cells from different groups; (E) Western blot analysis of LEPR protein expression in KMBC cells from different groups. n = 3.

[0019] Figure 5 Effects of LEPR upregulation on CCA cell proliferation. (A) CCK-8 assay to examine the effect of LEPR overexpression on SK-CHA-1 cell proliferation; (B) CCK-8 assay to examine the effect of LEPR overexpression on KMBC cell proliferation; (C) Plate-based colony formation assay to examine the effect of LEPR overexpression on SK-CHA-1 cell proliferation; (D) Plate-based colony formation assay to examine the effect of LEPR overexpression on KMBC cell proliferation. n = 3.

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

[0021] Figure 7 Effects of LEPR overexpression on the migration and invasion of SK-CHA-1 and KMBC cells. (A) Scratch wound assay to examine the effect of LEPR overexpression on the migration ability of SK-CHA-1 cells; (B) Scratch wound assay to examine the effect of LEPR overexpression on the migration ability of KMBC cells. n = 3. (C) Effects of LEPR overexpression on the migration and invasion ability of SK-CHA-1 cells; (D) Effects of LEPR overexpression on the migration and invasion ability of KMBC cells. n = 3.

[0022] In the attached figure:

[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 lentiviral overexpression plasmid. DETAILED DESCRIPTION

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

[0025] The present invention validated LEPR expression and its relationship with patient clinicopathological characteristics and survival in a cohort of researchers. Subsequently, the role of the LEPR gene in the development of CCA was confirmed from a cellular functional perspective. By constructing a stable cell line overexpressing the target gene LEPR, and then conducting cell proliferation and colony formation experiments, cell cycle, cell wound healing, cell migration, and cell invasion experiments, the results showed that upregulating LEPR expression levels inhibited the proliferation, migration, and invasion of CCA cells. This suggests that LEPR may serve as a target for CCA therapeutic drugs.

[0026] The present invention 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 assessment products.

[0027] In certain embodiments of the present invention, the cholangiocarcinoma is extrahepatic cholangiocarcinoma.

[0028] The present invention also provides use of the LEPR gene, LEPR protein or LEPR gene overexpression reagent in preparing a product for treating cholangiocarcinoma.

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

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

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

[0032] In certain embodiments of the present invention, the LEPR overexpression agent is selected from a nucleic acid construct comprising a LEPR gene or a virus having a LEPR gene integrated into its genome.

[0033] In certain embodiments of the present invention, the nucleic acid construct comprising the LEPR gene can be obtained by cloning a LEPR gene fragment (eg, the sequence shown in SEQ ID NO. 1) into a known vector.

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

[0035] Furthermore, the mammalian expression vector is selected from one or more of a LEPR gene adeno-associated virus vector, a LEPR gene lentivirus vector, a LEPR gene adenovirus vector or a LEPR gene retrovirus vector.

[0036] In an embodiment of the present invention, a lentiviral vector containing the LEPR gene is specifically constructed. The nucleotide sequence of the LEPR gene is shown in SEQ ID NO. 1. The LEPR gene lentiviral vector disclosed herein is obtained by cloning a LEPR gene fragment into a known vector, wherein the known vector is a lentiviral vector. The LEPR gene lentiviral vector is packaged into infectious viral particles, which are then used to infect cells, thereby overexpressing the LEPR gene.

[0037] In certain embodiments of the present invention, the virus into which the LEPR gene is integrated is selected from one or more of lentivirus, adenovirus, adeno-associated virus or retrovirus.

[0038] The bile duct cancer therapeutic 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 bile duct cancer therapeutic drugs prepared by using LEPR gene, LEPR protein or LEPR gene overexpression reagent include but are not limited to: nucleic acid molecules, antibody drugs, peptides, proteins, adeno-associated viruses or lentiviruses.

[0040] The dosage of the bile duct cancer therapeutic drug is sufficient to upregulate the transcription or translation of the LEPR gene, or to upregulate the expression or activity of the LEPR protein, so that the expression of the LEPR gene is upregulated by at least 50%, 80%, 90%, 95% or 99%.

[0041] LEPR overexpression reagents refer to substances that have an upregulating effect on LEPR. Substances that have an upregulating effect on LEPR include but are not limited to:

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

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

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

[0045] Promoting LEPR gene expression may specifically be promoting the transcription or translation of the LEPR gene. Specifically, it may mean increasing the LEPR gene transcription or expression by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by 70%, and most preferably by at least 90%.

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

[0047] 1) Inhibit the proliferation of bile duct cancer cells;

[0048] 2) Inhibit the clone formation of bile duct cancer cells;

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

[0050] 4) Induce cell cycle arrest.

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

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

[0053] The bile duct cancer treatment drug must include the LEPR gene, LEPR protein or LEPR overexpression reagent, and uses the LEPR gene, LEPR protein or LEPR overexpression reagent as the active ingredient for the above functions.

[0054] In the bile duct cancer treatment drug, the active ingredient that exerts the aforementioned function may be only the LEPR gene, LEPR protein or LEPR overexpression agent, or may contain other molecules that can exert similar functions.

[0055] The LEPR gene, LEPR protein or LEPR overexpression agent is the only active ingredient or one of the active ingredients of the drug for treating bile duct cancer.

[0056] The bile duct cancer treatment drug may be a single-component substance or a multi-component substance.

[0057] The product is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0058] The products include but are not limited to medicines, health products, food, etc.

[0059] The present invention also provides use of a substance that specifically binds to LEPR protein or its encoding gene in preparing a product for evaluating the prognosis of cholangiocarcinoma.

[0060] The TNM staging of extrahepatic bile duct cancer consists of T stage, N stage, and M stage. Each stage is further divided into different degrees. For example, the T stage is divided into T1, T2, T3, and T4 stages, the N stage is divided into N0, N1, and N2 stages, and the M stage is divided into M0 and M1 stages. The larger the value in each stage, the more serious the condition.

[0061] In certain embodiments of the present invention, the prognostic assessment product is used to detect the level of LEPR gene or protein.

[0062] The present invention also provides a method for treating cholangiocarcinoma, comprising administering a LEPR gene, a LEPR protein, or a LEPR overexpression agent to a subject.

[0063] The subject may be a mammal. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape, or a human.

[0064] The subject may be a patient suffering from bile duct cancer or an individual in whom prevention or alleviation of bile duct cancer is desired.

[0065] The LEPR gene, LEPR protein, or LEPR overexpressing agent can be administered to a subject before, during, or after receiving treatment for cholangiocarcinoma.

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

[0067] The expression of LEPR protein in the patient's body can be increased by directly administering LEPR protein or administering a product that promotes LEPR expression (such as an overexpression agent), or both.

[0068] The drug for treating bile duct cancer must include the LEPR gene, LEPR protein or LEPR overexpression agent, and uses the LEPR gene, LEPR protein or LEPR overexpression agent as the active ingredient for the aforementioned function.

[0069] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0070] A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, meaning it can be blended with it without significantly reducing the drug's effectiveness under normal circumstances. Pharmaceutically acceptable excipients include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, or lubricants. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; 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; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffers. These substances are used as needed to help stabilize the formulation or to enhance the activity or its bioavailability or to create an acceptable taste or flavor when taken orally.

[0071] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0072] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0073] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0074] The biological role of LEPR in CCA was studied through the following examples, and the results showed that upregulation of LEPR expression inhibited the proliferation, migration and invasion of CCA cells. The present invention verified the expression of LEPR and its relationship with the clinical pathological characteristics of patients and the survival of patients from the researcher cohort. Subsequently, the present invention confirmed the role of the LEPR gene in the occurrence and development of CCA from the perspective of cell function. By constructing a 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 compared with the lentivirus transfected control group. Subsequently, cell proliferation, migration, invasion and cell cycle were detected through cell functional experiments. The results showed that the degree of CCA cell proliferation 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 diagnostic and treatment methods for this gene can provide more options for the diagnosis and treatment of CCA patients.

[0075] Example 1 Differences in LEPR protein expression in CCA and ANT in the researcher cohort

[0076] This example is based on 60 retrospectively collected CCA patient tissues from the researcher cohort. IHC detection found that LEPR was significantly lower in CCA tissues than in control ANT tissues (P<0.001, Figure 1 AC). Western blot was used to detect the protein expression of LEPR in 5 paired fresh CCA and ANT surgical specimens. Postoperative fresh CCA tissues and their paired ANT tissues also confirmed that LEPR was significantly lower in CCA tissues than in control ANT tissues ( Figure 1 D).

[0077] Example 2: Exploring the relationship between LEPR expression and clinical pathological characteristics of CCA patients based on a cohort of researchers

[0078] To clarify the clinical value of LEPR in Chinese CCA patients, this study conducted a correlation analysis between LEPR expression levels and clinicopathological characteristics of CCA patients based on IHC results of 60 patients in the researcher cohort. The results showed that the lower the LEPR expression, the higher the T stage and pathological stage ( Figure 2 AE), which indicates that the lower the expression of LEPR, the more severe the CCA disease in patients.

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

[0080] This study analyzed the correlation between LEPR expression levels and CCA patient survival based on IHC results from a cohort of 60 researchers. Kaplan-Meier survival analysis showed that CCA patients with low LEPR expression had a shorter overall survival (OS) than those with high LEPR expression. Figure 3 A). Univariate COX regression analysis showed that T stage, pathological stage and LEPR expression level were predictive factors for the prognosis of CCA patients. Multivariate COX further confirmed that LEPR expression and T stage were independent predictive factors for the prognosis of CCA patients (all P < 0.05, Figure 3 B).

[0081] Example 4 Construction of a CCA cell model that stably upregulates LEPR

[0082] To further investigate the role of LEPR in CCA, this study used Western blot to detect the expression levels of LEPR in five CCA cell lines, 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 was low in the eCCA cell lines SK-CHA-1 and KMBC ( Figure 4 A). Therefore, SK-CHA-1 and KMBC cells were selected as tool cells for subsequent experiments. SK-CHA-1 cell models and KMBC cell models stably overexpressing LEPR were constructed, respectively, and the results were confirmed by qRT-PCR and Western blot.

[0083] (1) Construction of overexpression plasmid vector

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

[0085]

[0086]

[0087]

[0088] (2) Lentiviral packaging

[0089] Lentivirus packaging was completed by Shanghai JiKai Biotechnology Co., Ltd. The specific packaging process was carried out in accordance with the instructions of Shanghai JiKai Biotechnology Co., Ltd. The general process is as follows: a tool vector plasmid carrying the target gene or target sequence, a viral packaging helper plasmid Helper1.0, and a viral packaging helper plasmid Helper2.0 were used to co-transfect 293T cells. The virus was harvested 48-72 hours after transfection (i.e., the unpurified cell supernatant), concentrated and purified to obtain a high-titer lentivirus storage solution, and all indicators of the lentivirus were tested to meet the quality standards.

[0090] (3) Lentiviral infection

[0091] KMBC and SK-CHA-1 cell lines in good growth condition were digested and centrifuged and then inoculated into six-well plates. The transfection experiment was started when the cell confluence reached 30% and entered the logarithmic growth phase. The virus volume required for transfection was determined according to the multiplicity of infection (MOI) of the cells according to the instructions provided by GeneCare (virus volume added to each well = MOI * number of cells / virus titer).

[0092] (4) Transfection efficiency evaluation

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

[0094] The results are as follows Figure 4 As shown, the results showed that LEPR expression was low in eCCA cell lines SK-CHA-1 and KMBC ( 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 were constructed ( Figure 4 BC) and KMBC( Figure 4 DE) cell model and 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 assay: The method is as follows: take cells in excellent condition and digest them with trypsin, then adjust the cell suspension concentration to 2.5×10 3cells / 100μl and inoculated into 96-well plates (100μl per well). Place in a 37°C, 5% CO2 constant temperature incubator and incubate for 24 hours to allow complete attachment. At three time points, 1 / 2 / 3 days after intervention, remove the culture medium in the wells and add 10μl CCK-8 working solution (diluted 1:10 with basal 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 Preheat the M5 microplate reader to 37°C and set the dual-wavelength detection mode (450 nm detection wavelength / 630 nm reference wavelength) to measure the absorbance of each well. The OD value of the experimental group is converted to the relative proliferation rate (%) = (OD of the experimental group / OD of the control group) × 100%.

[0097] The results of CCK-8 assay showed that there was no significant difference in the proliferation ability of SK-CHA-1 cells in the LV-LEPR group compared with the LV-Control group on day 1, but after days 2 and 3, the proliferation of SK-CHA-1 cells in the LV-LEPR group decreased significantly (P<0.001) ( Figure 5 A). In KMBC cells, the proliferation of the LV-LEPR group was significantly inhibited at different detection points (P<0.001) ( Figure 5 B).

[0098] 2. Plate cloning assay: (1) Prepare single cell suspension and adjust the density to 3×10 2 cells / ml, inoculate 2 ml per well into a 6-well plate. Maintain a constant temperature environment at 37°C for 10-14 days, and replace the complete medium containing 10% FBS every 72 hours. When clones with a diameter >0.5 mm are visible to the naked eye, discard the medium and gently rinse three times with 4°C pre-cooled PBS. Add 4% paraformaldehyde to fix for 15 minutes and stain with 0.1% crystal violet (prepared in methanol) for 20 minutes. Rinse with double distilled water until the background is transparent, dry at room temperature, and collect images using an inverted microscope (4× objective lens). Image J software was used to analyze the clone formation rate: number of clones / number of seeded cells × 100% (threshold set to >50 cells / clone).

[0099] The results of the clone formation experiment showed that the number of clones formed by SK-CHA-1 and KMBC cells in the LV-LEPR group was significantly lower than that in the LV-Control group (P<0.001) ( Figure 5 CD).

[0100] 3. Flow cytometry was used to detect cell staging. The method was as follows: the required cells were digested and counted, and 5×10 5Place the cells to be tested in a centrifuge tube and perform centrifugation. After centrifugation for 4 minutes, remove the tube and decant the supernatant, excluding the cell pellet. Use a pipette to add an appropriate amount of PBS, then perform centrifugation again. After centrifugation, decant the supernatant. Add 1 mL of DNA staining solution to the tube and pipette to mix thoroughly. Then, use a small-range pipette to draw up 10 μL of permeabilization solution, vortex to mix thoroughly, and let it stand at room temperature. After 30 minutes, remove the tube, configure the appropriate flow cytometer program, and perform the test.

[0101] Flow cytometry results are as follows: Cell cycle analysis showed that LEPR overexpression led to a significant decrease in the proportion of SK-CHA-1 cells in the G2 / M phase (P<0.05) ( Figure 6 A), while in KMBC cells, LEPR overexpression led to a significant increase in the proportion of cells in S phase (P<0.05) ( Figure 6 B) These results indicate that LEPR upregulation can effectively inhibit the proliferation of CCA cells and induce cell cycle arrest.

[0102] 4. Cell scratch test: The method is as follows: the seeding density in 6-well plates is 5×10 5 cells / well, and when the degree of confluence reaches 90%, use a 200μl sterile pipette tip to scratch the surface of the well plate perpendicularly. Gently rinse with PBS three times to remove detached cells and replace with 2% FBS culture medium. The scratch width was recorded at three time points of 0 / 12 / 24 hours. The healing rate was calculated using the TScratch algorithm: (initial area-final area) / initial area×100%. The results of the scratch experiment showed that after 24 hours, the scratch healing rate of SK-CHA-1 and KMBC cells in the LV-LEPR group was significantly lower than that in the NC group, and the lateral migration inhibition of SK-CHA-1 cells was more significant (P<0.001)( Figure 7 A), while the migration inhibition of KMBC cells was smaller (P<0.05) ( Figure 7 B).

[0103] 5. Migration assay: Add 600 μl of complete culture medium to each lower chamber of the Transwell plate, place the sterile chamber on the lower chamber, and make sure there are no bubbles between the chamber and the lower chamber. Digest and count the desired cells (using basal culture medium) at a concentration of 1.5×10 cells per well. 4The cells were seeded at a density of 100 μg / cm2 on the upper layer of the chamber and cultured in a cell culture incubator for 24 hours. Remove the Transwell plate, take out the chamber, and fix the cells with 4% paraformaldehyde for 30 minutes. After rinsing with PBS to remove the paraformaldehyde, place in crystal violet and incubate in the dark for 30 minutes. Use tweezers to clamp the upper edge of the chamber, rinse repeatedly in PBS to remove the crystal violet and dry it. Place the chamber under a microscope, select 5 fields of view, take pictures and count. The experimental 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 upregulation of LEPR inhibited the vertical migration of cells ( Figure 7 CD).

[0104] 6. Invasion assay: The method is as follows: Diluted with serum-free medium at a ratio of 1:8, pre-plated in the upper chamber of a Transwell chamber (8 μm pore size) (50 μl / well), and polymerized at 37°C for 2 hours to form a basement membrane model. The upper chamber was loaded with 1.5×10 4 Cells were suspended in 150 μl of serum-free suspension, and 600 μl of chemotaxis medium containing 20% ​​FBS was injected into the lower chamber. After 24 hours of culture, cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.5% crystal violet to quantify the number of cells that passed through the membrane. The experimental results showed that similar to the results of the migration experiment, further confirming the inhibitory effect of LEPR upregulation on cell invasion ability ( Figure 7 CD).

[0105] These results show that upregulating LEPR expression can inhibit the proliferation, migration, and invasion of CCA cells. This suggests that LEPR can be used as a therapeutic target in the preparation or screening of drugs for the treatment of CCA, as well as in the preparation or screening of drugs that inhibit CCA proliferation, migration, and / or invasion.

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

Claims

1. Use of the LEPR gene or protein as a target in the preparation or screening of cholangiocarcinoma therapeutic products or prognosis assessment products.

2. Use of the LEPR gene, LEPR protein or LEPR gene overexpression reagent in the preparation of a product for the treatment of cholangiocarcinoma.

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

4. The use according to claim 2, characterized in that The LEPR gene is a LEPR gene fragment or a full-length LEPR gene sequence, or the LEPR protein is a complete LEPR protein or a protein fragment; or the LEPR protein is a natural LEPR protein or a recombinant LEPR protein.

5. The use according to claim 2, characterized in that The LEPR overexpression agent is selected from a nucleic acid construct containing the LEPR gene or a virus with the LEPR gene integrated into its genome; preferably, the nucleotide sequence of the LEPR gene is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that The LEPR gene nucleic acid construct is a mammalian expression vector; preferably, the mammalian expression vector is selected from one or more of a LEPR gene adeno-associated virus vector, a LEPR gene lentivirus vector, a LEPR gene adenovirus vector or a LEPR gene retrovirus vector.

7. The use according to claim 5, characterized in that The virus with the LEPR gene integrated into its genome is selected from one or more of lentivirus, adenovirus, adeno-associated virus or retrovirus.

8. Use of a LEPR gene, LEPR protein, or LEPR overexpression reagent in the preparation of a product having any of the following effects: 1) Inhibit the proliferation of bile duct cancer cells; 2) Inhibit the clone formation of cholangiocarcinoma cells; 3) Inhibit the invasion or migration of cholangiocarcinoma cells; 4) Induce cell cycle arrest.

9. A composition for treating bile duct cancer, wherein the effective substance comprises LEPR gene, LEPR protein or LEPR overexpression agent and a pharmaceutically acceptable carrier or excipient.

10. Use of a substance that specifically binds to LEPR protein or its encoding gene in the preparation of a product for the prognosis assessment of cholangiocarcinoma.

Images