Application of substance for inhibiting expression of oxytocin receptor in preparation of medicine for treating liver cancer

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of a substance for inhibiting expression of an oxytocin receptor in preparation of a medicine for treating liver cancer. The amino acid sequence of the oxytocin receptor is as shown in SEQ ID NO. 1. By reducing the expression of the oxytocin receptor, it is found that proliferation and migration of the cells can be well inhibited. Through an in-vivo nude mouse tumor-bearing experimental model, the result also shows that the in-vivo proliferation ability of liver cancer cells can be obviously weakened by inhibiting the oxytocin receptor OXTR. Cell experiments, animal experiments and clinical sample analysis prove that the oxytocin antagonist atosiban can significantly inhibit the progress of liver cancer cells.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and disease treatment, and particularly relates to use of a substance that inhibits the expression of oxytocin receptors in preparing a medicine for treating liver cancer. Background Art

[0002] Liver cancer is one of the most deadly malignant tumors in the world, with 900,000 new cases and about 830,000 deaths each year. Although most liver cancer patients can be treated with surgery, the overall survival rate of liver cancer patients is poor. Oxytocin receptor (OXTR) is a member of the G protein-coupled receptor family. G protein-coupled receptors (GPCRs) can sense a series of ligands in the extracellular environment and are also involved in the regulation of a series of physiological activities. However, recent studies have also shown that G protein-coupled receptors can participate in the growth and metastasis of certain tumors.

[0003] Atosiban is a specific antagonist of oxytocin receptors. It can interact with oxytocin receptors (OXTR). Its unique mode of action is that it can competitively bind to oxytocin receptors on the myometrium or decidua. Through this mechanism, atosiban can effectively inhibit the rise of intracellular calcium ions, thereby exerting its efficacy and can be used to treat premature delivery. Therefore, the clinical application of atosiban provides a new treatment strategy for premature patients, which is of great significance for protecting the health of mothers and infants.

[0004] Currently, oxytocin receptors can regulate the development of some tumors. However, there are currently no substances that inhibit the expression of oxytocin receptors used in products for the treatment of liver cancer. Summary of the invention

[0005] In order to solve the problem that there is no substance that inhibits the expression of oxytocin receptors in the prior art for use in products for treating liver cancer, a use of oxytocin receptors in the preparation of drugs for treating liver cancer is provided.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] Primary liver cancer is a common malignant tumor in my country. It is generally accompanied by extensive intrahepatic and extrahepatic metastasis, so the prognosis of patients is generally poor. Some factors, including significant changes in the epigenetic level between tumor cells and normal liver cells, and abnormal cell metabolism, also provide favorable conditions for the development of liver cancer. In addition, studies have found that there are complex and subtle interactions between many tumor cells and their surrounding environment. This interaction further promotes the malignant growth and changes of liver cancer. Moreover, in the context of extensive clinical and in-depth basic research, a variety of liver cancer prognostic markers have been conclusively verified. They provide important biomarkers for the prediction, evaluation and treatment of liver cancer. Despite continuous scientific research efforts, the early identification of liver cancer and the formulation of effective treatment strategies still face significant difficulties and challenges.

[0008] G protein-coupled receptors are the largest class of cell membrane receptors discovered so far. They play a vital role in organisms and are involved in most physiological activities and signal regulation processes. However, when the function of GPCRs is disordered, it may lead to a series of diseases. Including inflammation, immune diseases, cancer and other diseases. GPCRs not only occupy a core position in physiological activities, but are also one of the key targets in the field of drug research and development. According to statistics, drugs related to GPCRs account for 40% to 50% of the drugs on the market. In view of this, in-depth research on the structure and function of GPCRs will help us better understand its role in physiological and pathological processes. It is of great significance to explore new therapeutic targets for GPCRs-related diseases and the development of new drugs.

[0009] The human oxytocin receptor (OXTR) receptor is composed of 389 amino acids in total, and it also has 7 transmembrane domains. Therefore, it is a key molecule with a special polypeptide structure. Studies have shown that oxytocin receptors have a significant regulatory effect on animal reproduction, lactation, and energy metabolism. OXTR knockout mice show significant weight gain and late-onset obesity compared to wild-type mice. It is also accompanied by an increase in body temperature. Among them, the oxytocin receptor is also called the oxytocin receptor.

[0010] In the in-depth study of the present invention, we found that the expression level of oxytocin receptor in liver cancer tissue was significantly upregulated, and this upregulation phenomenon was closely related to the poor prognosis of liver cancer patients. Based on this, the purpose of the present invention is to provide a substance that inhibits the expression of oxytocin receptor in the preparation of a drug for treating liver cancer. This solves the defect that there is no application of oxytocin receptor in the treatment of liver cancer products.

[0011] The amino acid sequence of the oxytocin receptor is shown in SEQ ID NO. 1. The base sequence encoding the oxytocin receptor is shown in SEQ ID NO. 2.

[0012] Preferably, the substance that inhibits the expression of the oxytocin receptor comprises siRNA.

[0013] The siRNA is used to induce silencing of the oxytocin receptor OXTR in cultured liver cancer cells through chemically synthesized short interfering RNA technology.

[0014] The sequence of the siRNA is as follows:

[0015] The nucleotide sequence of siControl is shown in SEQ ID NO.3: UUCUCCGAACGUGUCAC GUTT.

[0016] The nucleotide sequence of siOXTR#1 is shown in SEQ ID NO.4: GGCCUUAUCAGCUU CAAGATT.

[0017] The nucleotide sequence of siOXTR#2 is shown in SEQ ID NO.5: CGGUCAAGAUGACUUU CAUTT.

[0018] The substance that inhibits the expression of the oxytocin receptor is also referred to as an oxytocin receptor expression inhibitor.

[0019] Preferably, the expression inhibitor of oxytocin receptor includes an antagonist of oxytocin receptor, including any one of atosiban, barusiban, retosiban, and SSR126768A free base. The antagonist has chemical affinity for oxytocin receptor but no efficacy, thereby inhibiting the effect of the agonist on the receptor.

[0020] Atosiban is an oxytocin receptor (OXTR) antagonist. To further explore the role of atosiban in liver cancer, we added atosiban stimulation to liver cancer cell lines. Experiments have shown that atosiban can significantly reduce the protein level of OXTR. Studies have confirmed that atosiban can effectively inhibit the proliferation and invasion of liver cancer cells, thus clarifying its role in inhibiting the progression of liver cancer. Therefore, as a drug that targets and blocks the oxytocin receptor pathway, atosiban has great potential to become a new potential target for liver cancer treatment.

[0021] Preferably, the oxytocin receptor expression inhibitor further comprises L-366682, SHR1653, and OT-Rantagonist 1. The inhibitor can reduce the expression of oxytocin receptor accordingly depending on the concentration.

[0022] Preferably, the drug has the oxytocin receptor expression inhibitor as an active ingredient, and is supplemented with a pharmaceutically acceptable carrier. More preferably, the drug has the oxytocin receptor antagonist as an active ingredient, and is supplemented with a pharmaceutically acceptable carrier.

[0023] Preferably, the dosage form of the drug includes any one of an injection dosage form, an oral liquid dosage form and a capsule dosage form.

[0024] Preferably, the carrier comprises any one or more of water, saline, buffer, glycerol and ethanol.

[0025] Preferably, the drug is a solution; the solution is prepared with the substance that inhibits the expression of the oxytocin receptor as an active ingredient and supplemented with a buffer solution.

[0026] The oral preparation is a solid or liquid preparation formed by combining the oxytocin receptor expression inhibitor with auxiliary materials and then taken orally, which is convenient to carry and take.

[0027] Injectable dosage forms include normal saline injection and sterile injection powder, which are quickly absorbed and effective.

[0028] The oral liquid dosage form is a liquid preparation prepared by using a conventional preparation method for oral liquid dosage forms to prepare the oxytocin receptor expression inhibitor. The drug is prepared in this dosage form, which is convenient to take.

[0029] Capsule dosage form is a solid preparation prepared by filling the oxytocin receptor expression inhibitor in a hollow hard capsule or sealing it in an elastic soft capsule. Specifically, it can be divided into hard capsules, soft capsules and enteric-coated capsules. Preparing the drug into a capsule can mask the bitter taste and special odor of the drug, and can also improve the stability and bioavailability of the drug, so that it can exert a greater therapeutic effect.

[0030] The present invention can prepare the oxytocin receptor expression inhibitor into the above pharmaceutical dosage form, but is not limited thereto. Any pharmaceutical dosage form that can be used to treat liver cancer can be prepared by using the oxytocin receptor expression inhibitor in the field of pharmaceutical preparations.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides the use of a substance that inhibits the expression of oxytocin receptors in the preparation of a drug for treating liver cancer. Preliminary experimental results of the present invention show that oxytocin receptors present a high expression pattern in liver cancer tissues, and this increased expression is not only closely related to the malignant progression of the tumor, but also has a direct correlation with the low survival rate of liver cancer patients. In order to further explore the biological function of oxytocin receptors, we knocked down oxytocin receptors in liver cancer cell lines. The results showed that when the activity of oxytocin receptors was inhibited, liver cancer cells showed obvious proliferation inhibition, accompanied by a significant weakening of migration ability. In order to verify the effectiveness of this finding, we used an in vivo animal model to evaluate the effect of silencing oxytocin receptors on liver cancer cells. The experimental results confirmed that in these tumors, the reduction in cell proliferation caused by silencing oxytocin receptors can effectively slow the development of tumors. We also proved that the oxytocin antagonist atosiban can significantly inhibit the progression of liver cancer cells through cell experiments, animal experiments and clinical sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The expression and prognosis of oxytocin receptor in liver cancer in the present invention, wherein: Figure 1 Figure A shows that the TCGA database shows that OXTR expression in patients with liver cancer is significantly increased (P<0.001); Figure 1 Figure B shows that high OXTR expression is associated with poor prognosis.

[0034] Figure 2 The knockout of oxytocin receptor in the present invention can affect the phenotype of liver cancer cells, wherein: Figure 2 Figures A to B in the figure show that SK-HEP1 and SNU449 liver cancer cells were transfected with siControl and siOXTR#1, #2, respectively, and the knockdown efficiency of OXTR was verified by western blotting after 24 hours; Figure 2 Figures C to H in the figure are transfected with siControl and siOXTR#1, #2 respectively. CCK8 cell proliferation assay and clone formation assay were used to verify that silencing OXTR in liver cancer SK-HEP1 cells and SNU449 cells can inhibit the proliferation of liver cancer cells. Figure 2 Figures I to L in the figure are transfected with siControl and siOXTR#1, #2 respectively. Cell scratch assay was used to verify that silencing OXTR in liver cancer SK-HEP1 cells and SNU449 cells can inhibit the migration ability of liver cancer cells; Figure 2 Figures M to O in the figure are subcutaneous tumor formation experiments that prove that inhibiting oxytocin receptors can inhibit the growth of liver cancer cells; among them, Figure 2 Figure M in the figure is a photo of a tumor in a nude mouse subcutaneous tumor-bearing experiment; Figure 2 Figure N in the figure is a comparison of tumor weight. The tumor was removed and weighed; Figure 2Figure O in the figure is a tumor growth curve. After tumor cells were implanted subcutaneously in nude mice, the length and width of the tumor were measured every week, and the tumor volume was calculated according to the formula Volume = (width)2×length / 2.

[0035] Figure 3 The oxytocin receptor antagonist atosiban used in the present invention can also affect the phenotype of liver cancer cells, wherein: Figure 3 Figures A to B in the figure are Western blotting experiments to detect the regulatory effect of atosiban on OXTR in liver cancer cells; Figure 3 Figures C to H in the figure show that the OXTR inhibitor Atosiban was used to treat cells at concentrations of 5μM and 10μM, respectively. CCK8 experiments and clone formation experiments verified that Atosiban can inhibit the proliferation of liver cancer cells in liver cancer SK-HEP1 cells and SNU449 cells; Figure 3 Figures I to L in the figure show that the OXTR inhibitor Atosiban was used to treat cells at concentrations of 5μM and 10μM, respectively. The cell scratch experiment verified that Atosiban can inhibit the migration ability of liver cancer cells in liver cancer SK-HEP1 cells and SNU449 cells; Figure 3 Figures M-O in the figure are subcutaneous tumor formation experiments that prove that the addition of atosiban can inhibit the growth rate of liver cancer cells in nude mice; Figure 3 Figure M in the figure is a photo of a tumor in a nude mouse subcutaneous tumor-bearing experiment; Figure 3 Figure N in the figure is a comparison of tumor weight. The tumor was removed and weighed; Figure 3 Figure O in the figure is a tumor growth curve. After tumor cells were implanted subcutaneously in nude mice, the length and width of the tumor were measured every week, and the tumor volume was calculated according to the formula Volume = (width)2×length / 2. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0037] The following are the experimental cells and experimental materials involved in the examples:

[0038] 1. Experimental Materials and Reagents

[0039] 2000 transfection kit: ThermoFisher Scientific; DMEM cell culture medium: Thermo, USA; Australian fetal bovine serum: Thermo, USA; trypsin: Thermo, USA;

[0040] Opti-MEM: Thermo, USA; Interferen: Thermo, USA; Penicillin-Streptomycin: Bio-Tech, China; WST-1 reagent: Roche, USA; SYBR Green: Roche, USA; Fludarabine: Selleck, USA; T25 cell culture flask: Corning, USA; 6-well cell culture plate: Corning, USA; 24-well cell culture plate: Corning, USA; 96-well cell culture plate: Corning, USA; 50 mL centrifuge tube: Corning, USA; 15 mL centrifuge tube: Corning, USA; 1.5 mL EP tube: Aixin, USA; 0.6 mL EP tube: Aisheng, USA; large pipette tip: Aisheng, USA; medium pipette tip: Aisheng, USA; small pipette tip: Aisheng, USA; disposable rubber-tipped dropper: NEST, China; sodium chloride: Tianjin De'en Chemical Co., Ltd., China; potassium chloride: Tianjin De'en Chemical Co., Ltd., China; Tween: Shanghai Sinopharm Chemical Reagent Co., Ltd.; methanol: Shanghai Sinopharm Chemical Reagent Co., Ltd.; ethanol; Tianjin De'en Chemical Co., Ltd., China; isopropanol; Shanghai Sinopharm Chemical Reagent Co., Ltd.; anhydrous ethanol; Tianjin De'en Chemical Co., Ltd., China; colored pre-stained protein molecular weight standard; Beyotime Biotechnology, China; OXTR antibody; SANTACRUZ, USA; β-Actin antibody; CST, UK; total RNA extraction kit; TianGen, China; PVDF membrane; GenScript, USA; ECL luminescent solution; GE Healthcare, UK; SDS-PAGE gel rapid preparation kit; Beyotime Biotechnology, China; Marker; Beyotime Biotechnology, China.

[0041] 2. Main instruments and equipment

[0042] CO2 cell culture incubator; Thermo scientific, USA; microscope; Nikon, model YS100 Japan; ordinary refrigerator (4℃, -20℃); Xinfei refrigerator, Xinxiang, China; -80℃ refrigerator; Eppendorf China Co., Ltd., Germany; microplate reader; Thermo miltiskan MK3, USA; ice maker; SANYO, SIM-F140AY65 Japan; centrifuge; ThermoScientific, USA; electrophoresis instrument; BIO-RAD Mini-Protean tetra system; digital display constant temperature water bath; Changzhou Zhiborui Instrument Manufacturing Co., Ltd.; horizontal shaker; China Jintan Guosheng Experimental Instrument Factory, China; clean bench; Thermoscientific, USA; 37℃ oven; Shanghai Fuma Experimental Equipment Co., Ltd.; real-time quantitative fluorescence PCR instrument; AppliedBiosystems, USA; ultrapure water system; Direct-Q 3UV, ultrapure; exposure machine; Applied Biosystems, USA.

[0043] 3. Cells

[0044] SK-HEP1 cells and SNU449 cells were kindly donated by Karolinska University, Sweden.

[0045] 4. Experimental Animals

[0046] Six-week-old BALB / c nude mice were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.

[0047] 5. Oxytocin receptor (OXTR) is composed of 389 amino acids and has 7 transmembrane domains. OXTR has a significant regulatory effect on animal reproduction, lactation, and energy metabolism. Studies have shown that OXTR knockout mice have significant weight gain and late-onset obesity compared to wild-type mice, accompanied by increased body temperature.

[0048] Among them, the English abbreviation of oxytocin receptor is OXTR.

[0049] The amino acid sequence of the oxytocin receptor is shown in SEQ ID NO.1;

[0050] MEGALAANWSAEAANASAAPPGAEGNRTAGPPRRNEALARVEVAVLCL ILLLALSGNACVLLALRTTRQKHSRLFFFMKHLSIADLVVAVFQVLPQLLWDITFRFYGPDLLCRLVKYLQVVGMFASTYLLLLMSLDRCLAICQPLRSLRRRTDRLAVLATWLGCLVASAPQVHIFSLREVADGVFDCWAVFIQPWGPKAYITWITLAVYIVPVIVLAACYGLISFKIWQNLRLKTAAAAAAEAPEGAAAGDGGRVALARVSSVKLISKAKIRTVKMTFIIVLAFIVCWTPFFFVQMWSVWDANAPKEASAFIIVMLLASLNSCCNPWIYMLFTGHLFHELVQRFLCCSASYLKGRRLGETSASKKSNSSSFVLSHRSSSQRSCSQPSTA。

[0051] The base sequence encoding the oxytocin receptor is shown in SEQ ID NO.2:

[0052]

[0053] Example 1

[0054] 1. Methods

[0055] 1. Cell culture

[0056] The liver cancer cell lines used in this experiment were SK-HEP1 cells and SNU449 cells.

[0057] The SK-HEP1 cell culture medium is a high-glucose DMEM medium with 10% fetal bovine serum imported from Australia and 1% penicillin-streptomycin added to the culture medium, and the SNU449 cell culture medium is an RPMI-1640 medium with 10% fetal bovine serum imported from Australia and 1% penicillin-streptomycin (Biyuntian) added to the culture medium.

[0058] The cell culture process was carried out strictly in accordance with sterile operation, and the cell morphology and quantity were observed daily. At the same time, it was necessary to replace the DMEM medium or RPMI-1640 medium with cells in time.

[0059] The culture conditions for SK-HEP1 cells in DMEM medium were 37° C. and 5% CO 2 in a cell culture incubator, and the culture conditions for SNU449 cells in RPMI-1640 medium were 5% CO 2 in a cell culture incubator.

[0060] 2. siRNA transfection

[0061] The siRNA transfection system designed in this experiment is: 1.5 μL of transfection reagent RNA mix corresponds to 1 μL of siRNA. Each well of a 12-well plate contains 100 μL of transfection system.

[0062] Among them, the nucleotide sequence of siControl is shown in SEQ ID NO.3: UUCUCCGAACGUG UCACGUTT; the nucleotide sequence of siOXTR#1 is shown in SEQ ID NO.4: GGCCUUAUC AGCUUCAAGATT; the nucleotide sequence of siOXTR#2 is shown in SEQ ID NO.5: CGGUCA AGAUGACUUUCAUTT.

[0063] The detailed steps of transfection are as follows:

[0064] (1) Take out two 1.5 mL clean and sterile EP tubes prepared in advance, and then use a marker pen to mark them as tube A and tube B for easy identification and operation. First, add 50 μL Opti-MEM and 1.5 μL RNAmix to tube A, and then add 50 μL Opti-MEM and 1 μL siRNA to tube B, mix thoroughly, and leave at room temperature for 5 min.

[0065] (2) After 5 minutes of standing, gently add the liquid in tube B to tube A using a pipette. After this step is completed, place it at room temperature and wait for another 15 minutes. This period of time is enough to ensure that all the reaction substances can fully fuse and form a stable transfection signal.

[0066] (3) During the waiting process, change the cell medium, remove the old culture medium, and use a pipette to remove all the culture medium in the wells, add new 1640 culture medium, wait for tube A and tube B to be fully mixed, then take 100 μL of the prepared siRNA transfection system and add it to the 12-well plate, gently shake the plate to ensure that all siRNA is evenly distributed in each well, and then place the well plate in a cell culture incubator for 48 hours.

[0067] 3. Extraction of total RNA from cells

[0068] The purpose of this experiment is to extract total RNA from cells using the Trizol method, which is widely recognized and applied in the field of molecular biology. It can effectively isolate high-quality RNA from cells. The following are the specific steps:

[0069] (1) Before starting the experiment, it is necessary to ensure that the laboratory environment meets the sterility requirements, so the entire experiment is carried out in a fume hood. Prepare the reagents in advance: chloroform, isopropanol, 75% ethanol by volume, and 1.5mL clean sterile EP tubes. Remember to wear gloves and masks. Take out the 12-well plate of SK-HEP1 cells and SNU449 cells to be extracted, discard the culture medium, and wash each well in the 12-well plate with PBS. After washing, add 400μL of Trizol solution to each well, and pay attention to whether the cells are completely broken. After the cells are completely lysed, transfer the liquid in the well to another clean sterile EP tube.

[0070] (2) Next, add 80 μL of chloroform to each clean sterile EP tube. Vortex for about 15 seconds until the liquid in the EP tube becomes milky. Let stand at room temperature for 5 minutes, then centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0071] (3) After centrifugation, use a pipette to gently pipette 160 μL of the upper clear aqueous phase into an EP tube.

[0072] (4) After the above steps are completed, add isopropanol in an amount equal to the volume of the upper RNA aqueous phase. When the two liquids are thoroughly mixed, the sample needs to be left to stand at 4°C for 10 minutes. This step is to better mix the liquids. Then centrifuge at low speed for 10 minutes.

[0073] (5) After centrifugation, carefully remove the supernatant with a pipette and add 70% anhydrous ethanol to the EP tube to wash the precipitate. Let the sample stand at room temperature for 5 minutes, then centrifuge it again at 12,000 rpm at 4°C for 5 minutes in a centrifuge and discard the supernatant.

[0074] (6) Place the EP tube on an ice box, open the lid and let it sit for 5 minutes to allow the precipitate to completely dissolve.

[0075] (7) Finally, add 30 μL of ddH2O to dissolve the precipitate, and use a pipette to mix well to obtain an RNA solution. The total RNA concentration in the extract is measured by ultraviolet spectroscopy. After the concentration is measured, mark it for later use.

[0076] Among them, the steps of determining the total RNA concentration by ultraviolet spectroscopy are to use a pipette to draw 2 μL of RNA solution, add it to a specific measurement position, measure the A260 and A280 of the extracted RNA sample, and calculate the formula for the RNA concentration in the solution: RNA sample concentration (μg / mL) = A260×40×dilution factor.

[0077] 4. Real-time quantitative fluorescence PCR (qRT-PCR)

[0078] (1) After extracting total RNA from SK-HEP1 cells and SNU449 cells, a reverse transcription system can be prepared according to the components required in the reverse transcription system table in Table 1, and then reverse transcription can be performed.

[0079] Table 1 Reverse transcription 10 μL system

[0080] Reagents 10μL system <![CDATA[5x PrimeScript TM RT Master Mix]]> 2μL RNA 2μL RNase-Free Water 6μL

[0081] Note: The preparation should be done on ice.

[0082] (2) Configure the qRT-PCR system according to the requirements of Table 2.

[0083] Table 2qRT-PCR system

[0084]

[0085]

[0086] Note: The preparation should be done on ice.

[0087] (3) After the system is prepared, add it to a 96-well plate that matches the machine, add 10 μL to each well, and repeat the process to add it to three wells.

[0088] (4) Turn on the computer and set the program on the machine: pre-denaturation: 95°C, 5 min; cycle reaction: 95°C, 30 s; 55°C, 30 s; 72°C, 30 s; a total of 40 cycles. Finally, copy the results and calculate the results.

[0089] 5. Cell proliferation assay (CCK8)

[0090] (1) Cell plating: SK-HEP1 cells and SNU449 cells used in this experimental design were plated into two clean and sterile well plates respectively.

[0091] (2) When the cells in the plate are attached to the wall and the confluency reaches 30%, they are transfected with siControl / siOXTR respectively. The specific transfection method is the same as above.

[0092] (3) Cell counting after transfection: 24 hours after transfection, SK-HEP1 cells and SNU449 cells were digested with 1 mL of trypsin per well to obtain a digested and resuspended cell suspension with a concentration of 100,000 / mL. 10 μL of the digested and resuspended cell suspension was pipetted into a counting plate for cell counting.

[0093] (4) 96-well plate: To prepare for further experimental operations, first dilute the digested and resuspended cell suspension to a concentration of 50,000 / mL. Next, accurately add 100 μL of the diluted cell suspension to each pre-set 96-well plate. In order to more accurately control the experimental conditions, each plate needs to be plated with 3 replica wells, a total of 3 96-well plates, marked with different time points, and finally add a blank control well to each plate (only add an equal volume of culture medium). Finally, use PBS to surround the sample wells.

[0094] (5) Absorbance measurement: Before the absorbance test, 10 μL of CCK8 reagent needs to be accurately pipetted into the sample well and the blank control well, and then placed in a cell culture incubator for 1 hour. Next, a microplate reader is used to accurately measure the single wavelength of 450 nm for each well in the sample.

[0095] (6) According to the same method, at the same time every day, adopt the same method and process to measure the 96-well plate on the first day, the third day, and the fifth day respectively, and obtain the absorbance values ​​at different times.

[0096] (7) After the measurement, the obtained data is statistically analyzed and calculated.

[0097] 6. Protein Imprinting Technology

[0098] (1) Prepare SDS-PAGE gel: First, take two cleaned glass plates for gel preparation. Before starting the operation, the lower ends of the two plates must be aligned to prevent liquid leakage during the gel preparation process. Then, firmly fix the two glass plates to the gel preparation rack. Next, prepare the separation gel solution. This step is configured according to Table 3.

[0099] Table 38% separation gel

[0100] Reagents system Distilled water 6.9mL 30% acryl-bisacrylamidemix 3.9mL 1.5M Tris, pH = 8.8 3.9mL 10% SDS 150μL 10% APS, ammonium persulfate 150μL TEMED 9μL

[0101] After the solution is prepared, mix it as much as possible and add it to the glass plate immediately. When pouring the glue, the glue should be injected slowly and evenly from left to right. When about 7mL of glue is successfully injected into the glass plate, the injection can be stopped. Next, take 1mL of 75% ethanol solution and add it to each glass plate, and then let it stand for 30 minutes. After waiting for the upper layer of glue to solidify, start to prepare 5% concentrated glue according to the formula provided in Table 4. This step requires the same operating process as when preparing the upper layer of glue. At the same time, use the same method to inject the glue into the plate, and add about 3mL to each piece of glue with a pipette. Next, take out the required clean 15-hole comb and gently insert it into the concentrated glue. Try to keep the comb level in this step and don't have bubbles, otherwise it will affect the quality of the final glue. After the configuration is completed, let it stand for 30 minutes again.

[0102] Table 45% concentrated gel

[0103]

[0104]

[0105] (2) Sample loading: First, fix the prepared 5% concentrated gel on the electrophoresis stand. Then, carefully fill the inner tank with electrophoresis solution, and add 2 / 3 of the volume of electrophoresis solution to the outer tank. In order to prevent excessive heat generation during electrophoresis from affecting the gel running results, a frozen ice pack is generally placed. Then, gently pull out the comb in the concentrated gel, and add the pre-prepared SK-HEP1 cells and SNU449 protein samples to the gel wells in the order of control group, experimental group 1, and experimental group 2, respectively, with 10 μL of protein sample per well, and record the sample loading order.

[0106] (3) Electrophoresis: Turn on the electrophoresis machine and run the gel at a constant voltage of 80 V. After the protein sample runs to the lower gel, adjust the voltage to 120 V. Stop the electrophoresis when the smallest band of the marker approaches the bottom layer of the glass plate.

[0107] (4) Transfer: The PVDF membrane used in the experiment needs to be activated with methanol in advance. Then take a clean tray, add sufficient transfer solution to the container, and then immerse the transfer clip in it. After the transfer clip is properly placed, pour the transfer solution into the transfer tank to ensure that the transfer solution completely covers the transfer clip. Finally, because a lot of heat will be generated during the transfer, an ice pack can be put in, so as to achieve a good transfer effect. Turn on the transfer machine, adjust it to 300mA, 70min, and transfer at a constant flow.

[0108] (5) Milk blocking: After the transfer is completed, cut off a corner of the membrane with scissors and mark it. Then add 5 mL of skim milk to the box containing the membrane for blocking. The blocking time is 1 hour.

[0109] Among them, skimmed milk was purchased from Biyuntian (P0216).

[0110] (6) Primary antibody incubation: After the skim milk blocking reaches the specified time, the membrane is washed thoroughly with PBST solution, and then the primary antibody (antibody: primary antibody dilution = 1:1000) is added to the box and blocked on a shaker at room temperature for 4 h.

[0111] (7) Washing the membrane: After the time is up, make sure the primary antibody solution is promptly returned to the original tube to avoid waste. Then use PBST to wash the membrane thoroughly to remove unbound antibodies. Wash once every 10 minutes for a total of three times.

[0112] (8) Secondary antibody incubation: After washing the membrane, incubate with secondary antibody (secondary antibody: skim milk = 1:5000) and block for 1 h.

[0113] (9) Washing the membrane: Wash with PBST for 10 min each time, for three times.

[0114] (10) Development: Prepare the developer and use a developing machine for development.

[0115] (11) Copy and organize the results.

[0116] 7. Cell clone formation experiment

[0117] (1) Cell plating: The SK-HEP1 cells and SNU449 cells used in this experiment were plated into 12-well plates respectively.

[0118] (2) After the SK-HEP1 cells and SNU449 cells in the plate have adhered to the wall and the confluency has reached about 30%, they are transfected with siControl / siOXTR respectively. The specific transfection method is the same as above.

[0119] (3) 24 hours after transfection, SK-HEP1 cells and SNU449 cells were digested with 1 ml of trypsin per well to obtain a cell suspension with a concentration of 100,000 / mL. 10 μL of the cell suspension was pipetted into a counting plate for cell counting.

[0120] (4) Take out the used 6-well plate, add 2 mL of 1640 complete medium, and then inoculate the counted digested and resuspended cell suspension into the 6-well plate so that the number of cells inoculated in each well is about 600. Next, place the 6-well plate in a cell culture incubator at 37°C and culture for about 14 days. During this period, the medium of the well plate can be changed every four days.

[0121] (6) Fixation: After 14 days of culture, first discard the culture medium with a pipette. Then, wash each well of each well plate with PBS. Next, add 1 mL of paraformaldehyde fixative to each well of each 6-well plate to ensure that the cells are fully fixed. The fixation time is 15 minutes.

[0122] (7) Staining: After the time is up, the fixative solution is discarded with a pipette and each well in the well plate is washed again. Subsequently, 1 mL of crystal violet staining solution is added to each well to stain the cells evenly.

[0123] (8) After 15 minutes of staining, gently discard the staining solution with a pipette. Then, wash with PBS several times, air-dry appropriately, take pictures and store them in a well-lit place. After taking pictures, use ImageJ to count and average the numbers for quantitative research.

[0124] 8. Scratch test

[0125] (1) Cell plating: SK-HEP1 cells and SNU449 cells were plated into 6-well plates respectively.

[0126] (2) When the cells in the plate are attached to the wall and the confluency reaches about 30%, they are transfected with siControl / siOXTR respectively. The specific transfection method is the same as above.

[0127] (3) Scratching: When the cells are spread all over the well plate, we use a ruler as an auxiliary tool to ensure the accuracy of the operation. Then, take out a 200μL clean and sterile pipette tip in the direction perpendicular to the edge of the 6-well plate and gently draw two straight lines in each well. During the entire scratching process, we always maintain uniform force to ensure the consistency and accuracy of the scratching.

[0128] (4) Washing: In the wells treated with scratches, carefully wash the scratched wells three times with PBS, and then add 2 mL of freshly prepared cell culture medium.

[0129] (5) Photography: After washing the cells, observe them under a microscope and take photos at different magnifications. The photo taken at 0 h serves as the control group. After that, place the 6-well plate in a cell culture incubator and take out the 6-well plate at 24 h and 48 h, respectively. Take photos of the scratches at the same position under a microscope.

[0130] (6) After taking the photos, ImageJ was used to analyze and plot the changes in scratch width at different time periods to reflect the cell migration distance.

[0131] 9. Mouse tumor experiment

[0132] The present invention follows the strict guidelines of the Animal Protection Committee and carefully designs the experimental protocol for mice. These experimental animals are 6-week-old BALB / c Nude mice, and are randomly assigned to various experimental groups to ensure that each group contains 6 mice. The specific experimental groups are control group, experimental group 1, and experimental group 2.

[0133] Among them, the shControl and shOXTR stable transfected lines of the liver cancer SNU449 cell line were digested with trypsin, resuspended in 1 mL of cell culture medium, and divided into three groups and implanted subcutaneously in BALB / c nude mice. After mixing the tumor cells and matrix gel at a ratio of 1:1, 1x10 6 We used a stable transfected cell line of SNU449 cells to observe the tumor formation in mice. After tumor formation, the tumor size was detected every day. The volume of the tumor was calculated using the formula Volume = (width)2 × length / 2 to compare the growth of the three groups of tumors. When the average longest diameter of the tumor of any group of mice reached 2 cm, in the final stage of the experiment, we used cervical dislocation (quickly twisting the cervical vertebra of the mouse by hand or tools, causing spinal cord injury and death of the mouse) to end the life of the mouse, and then performed laparotomy on the mouse to remove the tumor. The removed tumor was carefully photographed to record its appearance characteristics, and then the volume and weight were accurately measured to ensure the accuracy and completeness of the experimental data. At the set statistical significance level (P < 0.05), we determined whether these changes were statistically significant, so that the reliability of the experimental results could be scientifically evaluated.

[0134] 10. Statistical methods

[0135] The experimental data were analyzed in depth by GraphPad Prism 7 and Excel software, and the results were presented in the form of mean ± standard deviation (x ± s). When comparing the data of different groups, we used the paired t-test statistical method. *P < 0.05, **P < 0.01, ***P < 0.001.

[0136] 2. Results

[0137] 1. Oxytocin receptor OXTR is expressed at an elevated level in liver cancer and is associated with a poor prognosis in patients with liver cancer

[0138] To explore the role of oxytocin receptors in the development of liver cancer, we took a systematic approach. First, we conducted a preliminary analysis of normal tissues and tumor tissues in the TCGA database. Figure 1 shown.

[0139] Depend on Figure 1 The results showed that compared with normal liver tissue, the expression level of oxytocin receptor OXTR in liver cancer tissue showed a significant increase trend, such as Figure 1 In addition, we also found that high expression of OXTR in liver cancer patients has a significant negative correlation with poor prognosis, as shown in Figure A. Figure 1 As shown in Figure B.

[0140] 2. Inhibition of oxytocin receptors can inhibit the phenotype of liver cancer cells

[0141] To verify whether OXTR expression can affect the phenotype of liver cancer cells, we silenced OXTR in SK-HEP1 cells and SNU449 cells. After 24 hours of transfection, we extracted protein samples from these two cell lines and then used protein immunoblotting to demonstrate the knockdown efficiency of OXTR. Figure 2 shown.

[0142] Depend on Figure 2 The experimental results showed that the knockdown efficiency of OXTR was confirmed in both SK-HEP1 cells and SNU449 cells. Figure 2 As shown in Figures A to B.

[0143] Subsequently, we conducted CCK8 experiments and observed that after knocking out OXTR in liver cancer cells, their growth rate was significantly slowed down. Figure 2 As shown in Figures C to D in Figure 2, the cell clone formation experiment further confirmed this point, indicating that as the OXTR activity decreased, the clone formation ability of SK-HEP1 cells and SNU449 cells decreased significantly, as shown in Figure 2. Figure 2 Figures E to H in the figure.

[0144] The above results clearly indicate the important role of OXTR in the proliferation of liver cancer cells.

[0145] Furthermore, in order to verify whether knocking out OXTR can affect the migration ability of liver cancer cells. Through cell scratch experiments, the experimental results showed that knocking down OXTR can significantly inhibit the migration ability of SK-HEP1 cells and SNU449 cells. Figure 2 As shown in Figures I to L.

[0146] The above results show that in the in vitro liver cancer cell line model, we have proved that silencing OXTR can affect the phenotype of liver cancer cells. Next, we want to explore whether silencing OXTR can affect the progression of liver cancer in vivo? We constructed shOXTR and shControl stable cell lines and conducted a subcutaneous tumor formation experiment in nude mice. The experimental results are as follows Figure 2 shown.

[0147] Depend on Figure 2 The results showed that compared with the control group, inhibition of oxytocin receptors can significantly inhibit the growth of liver cancer cells. Figure 2 As shown in Figures M to O.

[0148] 3. Atosiban can block the development of liver cancer by effectively inhibiting the activity of oxytocin receptors

[0149] Relevant data show that Atosiban is a new type of artificial polypeptide compound synthesized by modifying the structure of oxytocin. We then added different concentrations of Atosiban to SK-HEP1 cells and SNU449 cell lines, and protein immunoblotting experiments proved that Atosiban can significantly reduce the protein level of OXTR, such as Figure 3 As shown in Figures A to B.

[0150] Next, in the further study, we used CCK8 assay to evaluate the effect of Atosiban on the proliferation of liver cancer cells. The results showed that after using Atosiban, the proliferation of SK-HEP1 cells and SNU449 cells was significantly decreased compared with the control group. Figure 3 As shown in Figures C to D in the figure. The cell clone formation experiment also proved that the addition of atosiban showed a significant inhibitory effect on the proliferation of liver cancer cells, as shown in Figures C to D in the figure. Figure 3 As shown in Figures E to H.

[0151] On this basis, we further conducted a key in vitro research method, the cell scratch test, and once again proved that the addition of Atosiban can also significantly inhibit the migration ability of SK-HEP1 cells and SNU449 cells. Figure 3 As shown in Figures I to L.

[0152] Subsequently, liver cancer cells were implanted subcutaneously in BALB / c nude mice. Tumor cells were mixed with matrix gel at a ratio of 1:1, and 1×10 6Each group had 6 cells. After the tumor was formed, the mice were given normal saline and atosiban injections through the tail vein every day. The tumor size was measured every day. The experimental results showed that after the nude mice were injected with atosiban, the growth of liver cancer cells was significantly inhibited. Figure 3 As shown in Figures M to O.

[0153] In summary, the use of the oxytocin receptor antagonist atosiban can significantly inhibit the progression of liver cancer.

[0154] Primary liver cancer is the most common malignant tumor in my country, but the early identification of liver cancer and the formulation of effective treatment strategies still face significant difficulties and challenges. The present invention explores the role of oxytocin receptor OXTR in human liver cancer through a series of carefully designed experiments. Preliminary experimental results reveal a shocking but far-reaching discovery: OXTR is significantly upregulated in multiple liver cancer tissues, and this phenomenon is closely related to the poor prognosis of patients. We further studied liver cancer cell lines and confirmed that silencing the expression of OXTR can significantly inhibit the proliferation and migration ability of cells, which are one of the key factors in the spread of cancer.

[0155] To verify the validity of this discovery in vivo, we used nude mice as an experimental model. By silencing OXTR in a transplanted tumor model, we observed encouraging results: the growth rate of liver cancer cells was significantly slowed down and the tumor volume was reduced accordingly. This suggests that OXTR is not only a potential therapeutic target, but may also provide new ideas for future drug development.

[0156] In addition, we also comprehensively evaluated the effect of the OXTR inhibitor Atosiban through cell experiments, animal experiments, and clinical sample analysis. These rigorous research methods ultimately proved that the oxytocin antagonist Atosiban has significant anti-liver cancer activity, which can effectively inhibit the progression of liver cancer cells and may become a powerful tool for the clinical treatment of liver cancer.

[0157] The present invention provides the use of a substance for inhibiting the expression of oxytocin receptors in preparing a drug for treating liver cancer, and provides a new method for treating liver cancer.

[0158] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0159] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of a substance that inhibits the expression of oxytocin receptors in the preparation of a drug for treating liver cancer, characterized in that: The amino acid sequence of the oxytocin receptor is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The base sequence encoding the oxytocin receptor is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The substance that inhibits the expression of the oxytocin receptor includes siRNA; The siRNA includes any one or more of siControl, siOXTR#1 and siOXTR#2; The nucleotide sequence of the siControl is shown in SEQ ID NO.3; The nucleotide sequence of the siOXTR#1 is shown in SEQ ID NO.4; The nucleotide sequence of the siOXTR#2 is shown in SEQ ID NO.

5.

4. The use according to claim 3, characterized in that The substance that inhibits the expression of the oxytocin receptor also includes any one or more of atosiban, barusiban, retosiban and SSR126768A free base.

5. The use according to claim 3, characterized in that: The substance that inhibits the expression of the oxytocin receptor also includes any one or more of L-366682, SHR1653 and OT-R antagonist 1.

6. The use according to claim 1, characterized in that: The drug has a substance that inhibits the expression of the oxytocin receptor as an active ingredient and is supplemented with a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that The dosage form of the drug includes any one of an injection dosage form, an oral liquid dosage form and a capsule dosage form.

8. The use according to claim 6, characterized in that The carrier includes any one or more of water, saline, buffer, glycerol, and ethanol.

9. The use according to claim 7, characterized in that: The medicine is a solution; the solution is prepared by using a substance that inhibits the expression of the oxytocin receptor as an active ingredient and supplemented with a buffer solution.

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