Application of FTO in drug resistance treatment and prognosis prediction of ovarian cancer paclitaxel chemotherapy

Abstract

The invention relates to an application of FTO protein or gene as a diagnostic marker in preparation of an ovarian cancer paclitaxel chemotherapy drug resistance diagnostic reagent or kit. The invention also provides application of the FTO inhibitor in preparation of drugs for preventing or treating taxol chemotherapy drug resistance of ovarian cancer. The invention proves that the prognosis of the paclitaxel drug-resistant ovarian cancer can be predicted by detecting the FTO expression level in a human paclitaxel drug-resistant ovarian cancer patient for the first time, and prompts that the reagent for detecting the FTO expression level can be used for preparing the kit for predicting the prognosis of the ovarian cancer, so that a new way is provided for predicting the prognosis of the paclitaxel drug-resistant ovarian cancer. In conclusion, the reagent for detecting the FTO level can be used for preparing a reagent or a kit for predicting the ovarian cancer taxol drug resistance. At present, ovarian cancer is high in morbidity and difficult to treat, and the invention provides a new way for solving the clinical problem.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more particularly, to the use of FTO in the treatment of paclitaxel chemotherapy resistance and prognosis prediction in ovarian cancer. Background Art

[0002] Ovarian cancer (OC) ranks fifth among the causes of cancer-related deaths in women globally. Due to the lack of specific signs and symptoms in the early stages of the disease, ovarian cancer is usually diagnosed at a late stage, and peritoneal and distant metastases are common at the time of diagnosis. Therefore, its mortality rate ranks first among gynecological malignancies. The standard treatment for ovarian cancer is based on a combination of cytoreductive surgery and chemotherapy with taxanes and platinum drugs. Although ovarian cancer patients are usually initially sensitive to taxane and platinum chemotherapy and the treatment is effective, 70%-80% of patients will still relapse and eventually develop chemotherapy resistance. The median progression-free survival (PFS) of ovarian cancer relapse patients is estimated to be only 1-2 years. Therefore, great challenges exist in the diagnosis and treatment of ovarian cancer. Currently, paclitaxel remains the standard first-line chemotherapy drug for ovarian cancer, and the remission rate of patients in the initial treatment is high. However, due to the inherent high recurrence rate and drug resistance to chemotherapy drugs of ovarian cancer, the prognosis is poor, which greatly limits the clinical efficacy and application scope of chemotherapy and is also the main reason for the failure of ovarian cancer treatment.

[0003] Fat Mass and Obesity-associated Protein (FTO), as an m 6 A demethylase, regulates RNA function. FTO belongs to the Fe 2+ and αKG-dependent dioxygenase AlkB family proteins. In the pathogenesis and treatment of tumors, FTO plays a key biological role. FTO exhibits key biological functions in the pathogenesis and treatment processes of various tumors.

[0004] Chinese Patent Document CN111467497A discloses the application of FTO (fat and obesity related) protein as a target in the treatment of pressure overload-induced myocardial injury. This invention confirms that after the heart is stimulated by pressure overload, the down-regulation of FTO is an important cause of myocardial damage; in mice with pressure overload-induced myocardial injury overexpressing FTO, myocardial glucose uptake is significantly increased and cardiac function is significantly improved compared with those in the model group. It is thus concluded that overexpressing FTO can treat myocardial injury after pressure overload, and FTO is a drug target for treating pressure overload-induced myocardial injury. However, this invention does not involve its correlation with tumors, nor does it involve the detection and treatment of tumors. This invention also provides an FTO overexpression vector with remarkable efficacy. Existing studies have mostly demonstrated changes in the expression level of FTO in tumor cells or tissues, but have not proven whether FTO can be used as a biomarker to predict the prognosis of patients with paclitaxel-resistant ovarian cancer, and there is no relevant report in the prior art on using FTO level to predict the prognosis of ovarian cancer. Summary of the Invention

[0005] The purpose of this invention is to provide the use of FTO in the treatment of paclitaxel chemotherapy resistance and prognosis prediction of ovarian cancer in view of the deficiencies in the prior art.

[0006] In the first aspect, this invention provides the application of FTO protein or gene as a diagnostic biomarker in the preparation of a diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer.

[0007] As a preferred example, the kit includes primer pairs for detecting FTO gene or protein, and the primer pairs are as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] As a preferred example, the diagnostic samples for the diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer are tissues, serum, plasma, and urine.

[0009] In the second aspect, this invention provides the application of a reagent for detecting the content of FTO protein or gene in the preparation of a diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer.

[0010] As a preferred example, the reagent for detecting the content of FTO protein is selected from primer pairs specifically amplifying FTO; or probes specifically recognizing FTO or its transcript; or antibodies specifically against FTO protein.

[0011] As a preferred example, the kit includes primer pairs for detecting FTO gene or protein, and the primer pairs are as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0012] As a preferred example, the diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer comprises: nucleic acid extraction reagent; and / or polymerase chain reaction reagent; and / or protein immunoblotting reagent; and / or enzyme-linked immunosorbent assay reagent.

[0013] As a preferred example, the diagnostic samples of the diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer are tissues, serum, plasma and urine.

[0014] In a third aspect, the present invention provides the use of an inhibitor of FTO in the preparation of a drug for preventing or treating paclitaxel chemotherapy resistance in ovarian cancer.

[0015] The advantages of the present invention are as follows: The present invention for the first time demonstrates that the prognosis of paclitaxel-resistant ovarian cancer can be predicted by detecting the expression level of FTO in human paclitaxel-resistant ovarian cancer patients, suggesting that the reagent for detecting the expression level of FTO can be used to prepare a kit for predicting the prognosis of ovarian cancer. Therefore, a new way is provided for predicting the prognosis of paclitaxel-resistant ovarian cancer. In summary, the reagent for detecting the FTO level can be used to prepare a reagent or kit for predicting paclitaxel resistance in ovarian cancer. At present, the incidence of ovarian cancer is high and the treatment is difficult. The present invention provides a new way to solve this clinical problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To verify the expression level of FTO in two paclitaxel-resistant cell lines (OV3R-PTX and A2780-PTX) by western blotting and immunofluorescence staining; to verify the expression level of FTO in the tissues of ovarian cancer patients with recurrence after chemotherapy by immunohistochemical staining.

[0017] Figure 2 : Down-regulating the expression level of FTO can reduce the paclitaxel resistance of ovarian cancer. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The present invention will be further described below in conjunction with the specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0019] Example 1

[0020] Experimental method

[0021] 1. Extraction of total cellular protein and determination of protein concentration

[0022] (1) Prepare the equipment required for the experiment: ice, EP tubes, ordinary PBS buffer, cell scraper, SDS cell lysis buffer, PMSF protease inhibitor, PI phosphatase inhibitor, BCA protein assay kit, SDS loading buffer;

[0023] (2) Place the 6-well cell culture plate to be processed on ice, discard the used culture medium and wash it 3 times with PBS, and aspirate all the liquid;

[0024] (3) Prepare the cell lysis buffer according to the ratio of SDS:PMSF:PI = 100:1:1 and add it to the wells, shake well and place on ice for 30 min for sufficient lysis;

[0025] (4) Use a cell scraper to scrape the cell lysis buffer in the well plate (note to operate on ice), then aspirate it into a pre-cooled 1.5 mL EP tube, use an ultrasonic crusher on ice to break the cell structure to fully release the protein, centrifuge at 4 °C, 12000 g for 30 min;

[0026] (5) According to the BCA protein assay instructions, take the corresponding volume of protein supernatant for protein concentration determination;

[0027] (6) Take the corresponding volume of the remaining supernatant into a new pre-cooled 1.5 mL EP tube at 4 °C, add 4×loading buffer to the aspirated supernatant and dilute it to its working concentration, pipette and mix well, place in a 100 °C metal bath, shake and heat for 10 min, then place on ice for subsequent experiments or store in a -80 °C refrigerator.

[0028] 2. Protein immunoblotting experiment (Western blot)

[0029] (1) Gel preparation

[0030] 1) Prepare the required equipment: clean and dry tooth comb, thin / thick glass plates, gel-making rack, gel-making kit.

[0031] 2) Fix the glass plates on the gel-making rack, and prepare the upper and lower layer gel solutions of the required volume according to the instructions;

[0032] 3) Slowly inject the glass plates with the lower layer - upper layer gel in sequence at a constant speed, and quickly insert the tooth comb;

[0033] 4) Wait for about 30 minutes, and use the gel for electrophoresis after it solidifies or store it in a 4 °C refrigerator.

[0034] (2) Protein electrophoresis

[0035] 1) Prepare 1 L of fresh electrophoresis buffer: 100 mL of 10× Stock solution (30.3 g of Tris and 14.4 g of glycine made up to 1 L), 10 mL of 10% SDS solution, 890 mL of double-distilled water;

[0036] 2) Assemble the prepared gel into the electrophoresis device and add an appropriate amount of electrophoresis buffer to both the inner and outer tanks;

[0037] 3) Perform protein quantification and loading according to the experimental arrangement, and add protein Marker to the corresponding blank wells;

[0038] 4) Electrophorese at a constant voltage of 80 V until the protein Marker runs into the separating gel (about 30 min), then change to a constant voltage of 120 V

[0039] Continue electrophoresis and stop electrophoresis when the bromophenol blue reaches the appropriate level.

[0040] (3) Transfer membrane

[0041] 1) Prepare 1 L of transfer buffer: 100 mL of 10× Stock solution, 200 mL of methanol, 700 mL of double-distilled water;

[0042] 2) Prepare the transfer cassette, ice, ice box, filter paper, cut a suitable size of 0.45 μm / 0.2 μm PVDF

[0043] membrane and pour an appropriate amount of methanol to activate it;

[0044] 3) Place the moistened filter paper and the activated PVDF membrane on the transfer cassette, gently peel off the glass plate, cut the gel block, transfer it to the PVDF membrane, place it in the order of black gel and white membrane, clamp it and place it in the transfer device;

[0045] 4) Transfer at a constant current of 250 mA for about 90 minutes.

[0046] (4) Blocking

[0047] 1) Prepare 500 mL of TBST solution: 25 mL of 20× TBS, 500 μL of Tween-20, 475 mL

[0048] of double-distilled water;

[0049] 2) Prepare WB blocking solution (5% skim milk blocking solution) 50 mL: 2.5 g of skim milk made up to 50 mL of TBST solution;

[0050] 3) After membrane transfer, open the sandwich clamp, gently transfer the PVDF membrane to the blocking box (pay attention to the front and back of the membrane), place it on a horizontal shaker, and block at room temperature for 1 hour.

[0051] (5) Antibody incubation and exposure development

[0052] 4) Pipette the corresponding volume of antibody to the WB primary antibody diluent according to the recommended concentration in the antibody instruction manual. Immerse the blocked PVDF membrane fully in the prepared primary antibody solution and incubate overnight at 4°C.

[0053] 5) Recover the primary antibody solution, store it at -20°C, and wash the membrane with TBST for 0 min × 3 times.

[0054] 6) Prepare a secondary antibody solution with an appropriate concentration (1:5000 - 1:10000) using 5% skim milk blocking solution, cover and immerse the PVDF membrane, and incubate on a horizontal shaker at room temperature for 1 hour.

[0055] 7) Recover the secondary antibody solution, store it at -20°C, and wash the membrane with TBST for 10 min × 3 times.

[0056] 8) Prepare the ECL chemiluminescence solution according to the instruction manual with A liquid:B liquid = 1:1 by volume. Drop it evenly on the PVDF membrane and place it in the developer. Set appropriate exposure parameters for development. After completion, save the photo and statistically analyze the results.

[0057] 3. Immunohistochemical staining

[0058] After baking the tissue sections at 60°C to remove wax, rehydrate them successively in xylene - gradient ethanol and wash with PBS; perform citric acid antigen retrieval by microwave method (medium - high fire for 5 min → high fire for 2 min × 2); inactivate endogenous enzymes with 3% H2O2, permeabilize the membrane with 1% PBST, and block with 2.5% BSA at 37°C for 1 h; add 1:200 FTO primary antibody and incubate overnight at 4°C. After incubating the secondary antibody at room temperature for 30 min, develop with DAB (A:B = 19:1, monitor and terminate under the microscope); stain with hematoxylin for 5 min, differentiate with dilute hydrochloric acid, dehydrate with gradient ethanol, clear with xylene, and mount with neutral gum; perform double - blind microscopic examination and scoring (Table 1 - 1), and divide into high / low expression groups according to IHC score > 5 / ≤5.

[0059] Table 1 - 1 Immunohistochemical scoring table

[0060]

[0061]

[0062] 4. Immunofluorescent staining

[0063] (1) Discard the culture medium and wash 3 times with PBS.

[0064] (2) Fix the cells with 4% paraformaldehyde at room temperature for 20 min, wash 3 times with PBS, 5 min each time. Prepare a permeabilization solution of 0.5% Triton X with 5% BSA, cover the cell surface, and incubate at room temperature for 1 h.

[0065] (3) Discard the blocking solution, dilute the primary antibody at an appropriate concentration, place it in a humidified chamber, and incubate overnight at 4°C.

[0066] (4) The next day, warm it at 37°C for 0.5 h, wash 3 times with PBS, 5 min each time.

[0067] (5) Dilute the corresponding secondary antibody at an appropriate concentration, incubate in the dark at 37°C for 1 h, wash 3 times with PBS, 5 min each time. (6) Cover the cell surface with Hochest nuclear staining solution, stain in the dark at room temperature for 10 min.

[0068] (7) Observe with a laser confocal microscope and store in the dark at 4°C.

[0069] 5. Construct a stable FTO-knockdown cell line infected with lentivirus

[0070] (1) Hanyin Company synthesized the shRNA sequences targeting FTO as shown in Table 1-2. Co-transfect lentiviral packaging cells with the lentiviral vector and packaging plasmid, coat the virus, and collect the original virus solution.

[0071] Table 1-2 Primer sequences for constructing shFTO

[0072]

[0073] (2) Infection: Seed ovarian cancer paclitaxel-resistant cells OV3R-PTX and A2780-PTX cells in a six-well plate. When the cell density reaches 40%, perform the infection. Take out the virus solution, add 10 μg / mL of Polybrene at a ratio of 1 mL per well, and mix well. Then, discard the medium in the six-well plate and add the prepared infection solution to each well.

[0074] (3) Screening: Pre-seed the infected ovarian cancer paclitaxel-resistant cells OV3R-PTX and A2780-PTX cells in a 24-well plate, add puromycin at gradient concentrations to determine the puromycin concentration for screening. The screening concentration for OV3R-PTX is 10 mg / mL, and the screening concentration for A2780-PTX is 30 mg / mL. After 24 h of infection, change to the medium containing the corresponding screening concentration of puromycin and continue culturing for 48 h to preliminarily judge the infection efficiency based on the cell status.

[0075] (4) One week after screening, transfer the cells to a medium with a low concentration of puromycin, extract RNA to verify the overexpression efficiency, and transfer the cells into a T25 culture flask for subculture and cryopreservation to preserve the cell line.

[0076] 6. Nude mouse tumor-bearing experiment

[0077] (1) Forty-eight 3-4-week-old female BALB / c nude mice were purchased from Beijing Sbever Biotechnology Company and raised in an SPF-class animal room for 3 weeks. During this period, their body weights were measured, and the nude mice were randomly divided into four groups of 12 each, namely the shNC-Saline group, the sh-NC-PTX group, the shFTO-Saline

[0078] group, and the sh-FTO-PTX group, and ear tags were used for marking.

[0079] (2) OV3R-PTX cells with stably knocked down FTO and their control group cells were collected. After cell counting, cell suspensions were prepared using serum-free medium at a ratio of 1×10 6 cells / 100 μL per nude mouse. Subsequently, 1 mL of insulin syringe was used to subcutaneously inject the cell suspension into the right shoulder and back of the nude mice.

[0080] (3) After the injection, the nude mice were continuously raised normally for 2 weeks, during which the growth of tumors and the health status of the nude mice were recorded and observed.

[0081] (4) When the subcutaneous tumor volume reached approximately 40 mm 3 , according to the grouping of the nude mice, 100 μL of sterile saline and PTX injection (dose: 5 mg / kg, once every 3 days) were intraperitoneally injected respectively.

[0082] In the next 2 weeks, the body weights and tumor volumes of the nude mice were measured every two days.

[0083] (5) After 2 weeks of treatment, the nude mice were taken out of the SPF-class animal room. First, the nude mice were anesthetized with 10% sodium pentobarbital at a dose of 70 mg / kg

[0084] and, after complete anesthesia, the nude mice were arranged by group for photography.

[0085] (6) Subsequently, the nude mice were sacrificed by injecting an overdose of anesthetic, and tumor tissues were obtained. After measuring the tumor volume, the tissues were divided into two parts. One part was immersed in 4% paraformaldehyde for paraffin embedding; the other part was quickly frozen in liquid nitrogen and stored in a -80 °C refrigerator for subsequent RNA and protein extraction.

[0086] Results and Analysis

[0087] Please refer to Figure 1 , Figure 1(A) Western Blot was used to verify the expression of FTO in ovarian cancer parental cells and ovarian cancer PTX-resistant cells (A2780 / A2780-PTX, OVCAR-3 / OV3R-PTX). (B) The expression levels of FTO in primary OC tissues (n = 10) and recurrent OC tissues after treatment (n = 6). ns, no statistical significance; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (C) Immunofluorescence assay was used to detect the expression level and distribution of FTO in ovarian cancer parental cells and ovarian cancer PTX-resistant cells.

[0088] Figure 1 Western blotting and immunofluorescence staining were used to verify the expression level of FTO in two paclitaxel-resistant cell lines (OV3R-PTX and A2780-PTX); immunohistochemical staining was used to verify the expression level of FTO in tissues from patients with recurrent ovarian cancer after chemotherapy. As Figure 1 shown, compared with the paclitaxel-sensitive ovarian cancer parental cell lines (OVCAR-3 and A2780), at the protein expression level, FTO was highly expressed in human paclitaxel-resistant ovarian cancer cell lines (OV3R-PTX and A2780-PTX).

[0089] Please refer to Figure 2 , Figure 2 (A) Photographs of mouse tumors were taken. The first row was the control group + saline treatment, the second row was the control group + PTX treatment, the third row was the stable FTO knockdown group + saline treatment, and the fourth row was the stable FTO knockdown group + PTX treatment. There were 12 nude mice in each group. (B) During the PTX treatment, the changes in tumor volume of the mice were measured; Student's t-test was used to compare the differences in tumor volume among groups on the last day. n = 12. (C) HE staining, Ki67 and FTO immunohistochemical staining results of paraffin sections of tumor tissue samples from each group. The positive rate of FTO staining in each group is shown on the right, n = 12. ns, no statistical significance; ***, P < 0.001; ****, P < 0.0001.

[0090] As Figure 2 shown, downregulating the expression level of FTO can reduce the resistance of ovarian cancer to paclitaxel, inhibit the growth of ovarian cancer tumor volume, and improve the therapeutic effect of paclitaxel on ovarian cancer.

[0091] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of FTO protein or gene as a diagnostic marker in the preparation of a diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer.

2. Use of a reagent for detecting the content of FTO protein or gene in the preparation of a diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer.

3. The application according to claim 2, wherein The reagent for detecting the content of FTO protein is selected from primers for specifically amplifying FTO; or probes for specifically recognizing FTO or its transcript; or antibodies specifically against FTO protein.

4. The use according to any one of claims 1 to 3, characterized in that: The kit includes a primer pair for detecting FTO gene or protein, and the primer pair is shown as SEQ ID NO:1 and SEQ ID NO:

2.

5. The application according to claim 2, wherein The diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer includes: nucleic acid extraction reagent; and / or polymerase chain reaction reagent; and / or protein immunoblotting reagent; and / or enzyme-linked immunosorbent assay reagent.

6. The application according to any one of claims 1-5, characterized in that, The diagnostic samples for the diagnostic reagent or kit for paclitaxel chemotherapy resistance in ovarian cancer are tissues, serum, plasma and urine.

7. Use of an inhibitor of FTO in the preparation of a drug for preventing or treating paclitaxel chemotherapy resistance in ovarian cancer.

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