Combined medicine for treating breast cancer

Abstract

The invention provides a combined medicine for treating breast cancer, and belongs to the field of medicines. It is found that circSlc8A1 reduces the survival rate of breast cancer cells acted by adriamycin, enhances the chemosensitivity of the breast cancer cells, and meanwhile promotes the cell survival rate of myocardial cells induced by adriamycin chemotherapy. Animal experiments directly show that compared with breast cancer mice (not subjected to adriamycin treatment) only overexpressing circSlc8A1 and wild breast cancer mice (not subjected to adriamycin treatment) only subjected to adriamycin treatment, the tumor volume inhibition effect of the breast cancer mice overexpressing circSlc8A1 after being subjected to adriamycin treatment is greater than the sum of the breast cancer mice overexpressing circSlc8A1 and the wild breast cancer mice (not subjected to adriamycin treatment), and the tumor volume inhibition effect of the breast cancer mice overexpressing circSlc8A1 after being subjected to adriamycin treatment is better than the tumor volume inhibition effect of the wild breast cancer mice. The application proves that circSlc8A1 and adriamycin have a synergistic effect in breast cancer treatment, an unexpected technical effect is achieved, and a new choice is provided for clinical treatment of breast cancer and reduction of cardiotoxicity.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a combined medicine for treating breast cancer. Background Art

[0002] Breast cancer (BC) is the most common female malignancy worldwide and the leading cause of cancer death in women. The morbidity and mortality of breast cancer in my country rank first among female malignant diseases. Although comprehensive treatment including surgery, chemoradiotherapy and endocrine therapy has significantly improved the prognosis of breast cancer patients, drug resistance remains a difficult problem to be solved in the clinical treatment of breast cancer. Among them, anthracycline chemotherapy drugs have been widely used to treat various tumors including breast cancer. Doxorubicin (Dox, doxorubicin), as one of the most effective anthracycline chemotherapy drugs for breast cancer, is limited in its clinical application due to its severe cardiotoxicity. Therefore, exploring how to eliminate the chemotherapy resistance of malignant tumors including breast cancer and reduce the cardiotoxicity induced by chemotherapy drugs is still a hot issue that multiple disciplines are currently paying attention to.

[0003] Circular RNAs (circRNAs) are a class of small RNAs with closed circular structures. They are formed by exon skipping of pre-mRNA or reverse splicing of transcripts. They are not easily degraded by enzymes and are therefore highly stable in vivo. A large number of studies have shown that circular RNAs are involved in important physiological processes of tumor development and are closely related to tumor proliferation, invasion, and metastasis. Although some studies have suggested that circular RNAs are involved in multidrug resistance of tumors, the specific mechanism of their targeted resistance is still unclear. A few studies have pointed out that circRNAs in cancer also have the function of regulating chemotherapy-induced cardiotoxicity, but none of them have been studied in cancer treatment responses, and the specific mechanism is not yet fully understood. Therefore, it is of great significance to develop a new method to reduce cardiotoxicity without weakening the therapeutic effect of tumors or even enhancing chemotherapy sensitivity. Summary of the invention

[0004] The purpose of the present invention is to provide a combined medicine for treating breast cancer.

[0005] The present invention provides a combined drug, which contains a circular RNA and a chemotherapeutic drug for simultaneous or separate administration, or contains a reagent for overexpressing circular RNA and a chemotherapeutic drug for simultaneous or separate administration, wherein the circular RNA is circSlc8A1.

[0006] The present invention also provides a combined drug, which contains a reagent for overexpressing circular RNA and a chemotherapeutic drug for simultaneous or separate administration, wherein the circular RNA is circSlc8A1.

[0007] Furthermore, the chemotherapy drug is doxorubicin.

[0008] Furthermore, it also contains a pharmaceutically acceptable carrier.

[0009] Furthermore, the combined drug is an injectable preparation or an oral preparation.

[0010] Furthermore, the injection preparation is an intravenous injection preparation, an intraperitoneal injection preparation, an intramuscular injection preparation or a subcutaneous injection preparation;

[0011] The oral preparation is a decoction, an oral liquid, a granule, a capsule, a powder, a pill or a tablet.

[0012] The present invention also provides the use of the above-mentioned combined drug in preparing a drug for preventing and / or treating breast cancer.

[0013] Furthermore, the breast cancer is luminal breast cancer or triple-negative breast cancer.

[0014] The present invention also provides use of circular RNA in preparing a drug for alleviating cardiac toxicity induced by chemotherapeutic drugs, wherein the circular RNA is circSlc8A1.

[0015] Furthermore, the chemotherapy drug is doxorubicin.

[0016] The present invention also provides use of circular RNA in preparing a chemotherapeutic drug sensitizer, wherein the circular RNA is circSlc8A1.

[0017] Furthermore, the circSlc8A1 is obtained by overexpression.

[0018] Furthermore, the chemotherapy drug is doxorubicin.

[0019] The present invention found that circSlc8A1 reduced the survival rate of breast cancer cells treated with doxorubicin and enhanced the chemotherapy sensitivity of breast cancer cells, while promoting the cell survival rate of cardiomyocytes induced by doxorubicin chemotherapy. The present invention directly showed through animal experiments that compared with breast cancer mice that only overexpressed circSlc8A1 (not treated with doxorubicin) and wild-type breast cancer mice that only treated with doxorubicin (not overexpressing circSlc8A1), the effect of inhibiting tumor volume in breast cancer mice that overexpressed circSlc8A1 after doxorubicin treatment was greater than the sum of the effects of the above two, proving that circSlc8A1 and doxorubicin had a synergistic effect in the treatment of breast cancer, achieving unexpected technical effects, and providing a new option for clinical treatment of breast cancer and reduction of cardiac toxicity.

[0020] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0021] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Verification of the expression level of circSlc8A1: (A) RT-qPCR was used to verify the expression of circSlc8A1 in MCF7; (B) The expression level of circSlc8A1 was verified in AC16 before and after DOX treatment.

[0023] Figure 2 Effects of circSlc8A1 overexpression on doxorubicin sensitivity and cardiomyocyte toxicity in breast cancer cells: (A) Efficiency verification of circSlc8A1 overexpression in MCF7; (B) CCK-8 verification of changes in drug resistance after overexpression of circSlc8A1 in MCF7; (C) LDH verification of changes in cytotoxicity after overexpression of circSlc8A1 in MCF7; (D) Efficiency verification of circSlc8A1 overexpression in AC16; (E) CCK-8 verification of cell survival rate after overexpression of circSlc8A1 in AC16; (F) LDH verification of cytotoxicity after overexpression of circSlc8A1 in AC16.

[0024] Figure 3 The effect of doxorubicin treatment on tumors in circSlc8A1 transgenic mice before and after treatment: (A) The mammary fat pad tumor formation experiment of mouse breast cancer cell 4T1 is shown in the figure; (B) Tumor formation curve.

[0025] Figure 4 The effects of doxorubicin on the heart toxicity of circSlc8A1 transgenic mice: (A) Ultrasound assessment of cardiac function in transgenic mice; (B) Cardiac function indicators of transgenic mice: left ventricular pressure change rate; ejection fraction; left ventricular end-diastolic diameter; left ventricular end-systolic diameter. DETAILED DESCRIPTION

[0026] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0027] Example 1. Effects of circSlc8A1 and doxorubicin combined with anti-breast cancer and mitigation of doxorubicin-induced cardiotoxicity

[0028] 1. Experimental methods

[0029] (1) Cell experiments

[0030] 1) Normal breast cells (MCF-10A) were selected as the control group, and breast cancer cells (MCF7) were selected as the experimental group. Among them, MCF7 is a luminal breast cancer cell. The present invention first verified the expression level of circSlc8A1 in breast cancer cells (MCF7) and cardiomyocytes (AC16) using RT-qPCR.

[0031] 2) After overexpressing circSlc8A1 in breast cancer cells (MCF7) and cardiomyocytes (AC16) (the detailed process is shown in step (3)), different concentrations of doxorubicin (0 μM, 1 μM, 2 μM, 4 μM, 8 μM) were added for 24 hours. The cell viability was detected by CCK8, and the lactate dehydrogenase (LDH) release assay was used to detect the amount of lactate dehydrogenase released to evaluate the cytotoxicity.

[0032] (2) Real-time fluorescence quantitative PCR (RT-qPCR) detection of gene expression

[0033] 1) RNA reverse transcription

[0034]

[0035] Reaction conditions: 37°C, 15 min; 85°C, 5 s; stored at 4°C.

[0036] 2) RNA reverse transcription

[0037]

[0038] Reaction conditions: 95°C, 30s, 1 cycle; 95°C, 30s, 95°C, 5s, 60°C, 6s, 40 cycles; melting curve: 65.0°C to 95°C, temperature gradient of 0.5°C, time of 5s; Real-Time PCR analysis was performed using a fluorescent quantitative PCR instrument from Bio-Rad, USA.

[0039] Primer sequences:

[0040] circSlc8A1 forward primer F (SEQ ID No. 1): 5′-TGC CAG AGG TGG AGG GGA GGA T-3′;

[0041] circSlc8A1 reverse primer R (SEQ ID No. 2): 5′-ATC GCC GCA TGT TGT ACA TGA C-3′.

[0042] (3) Plasmid construction of circSlc8A1 overexpression vector and cell overexpression experiment

[0043] After MCF7 or AC16 cells were passaged and attached to the cell wall 24 hours later, the circSIc8A1 overexpression plasmid (circBase ID: hsa_circ_0005232) constructed based on the Bluescript skeleton was transfected into the experimental group cells. After 24 hours of culture, doxorubicin was added and treated for 24 hours for functional verification.

[0044] (4) Animal experiments

[0045] The construction method of transgenic mice overexpressing circSlc8A1 (named circSlc8A1(+ / +) mice) is as follows: during the cell proliferation process, eggs are collected and fertilized in vitro, and the exogenous gene (circBaseID: mmu_circ_0000823) is directly injected into the male pronucleus of the fertilized egg by a microinjection instrument to integrate the exogenous gene into the DNA, and the transgenic fertilized egg is implanted into the host mother mouse, and the transgenic mouse is developed into a transgenic mouse overexpressing circSlc8A1 after delivery. The primers for identifying transgenic mice are as follows:

[0046]

[0047] Then, the experimental groups:

[0048] (1) circSlc8A1+DOX group: Transgenic mice were subjected to mammary fat pad tumorigenesis experiments using mouse breast cancer cells 4T1. One week later, they were treated with doxorubicin at a dose of 6 mg / kg IP, once a week. Tumor and cardiac toxicity were evaluated 4 weeks later.

[0049] (2) circSlc8A1 group: Mouse breast cancer cell line 4T1 was used to perform a mammary fat pad tumor formation experiment on transgenic mice, and tumor and cardiac toxicity evaluation was performed at the same time as group (1).

[0050] (3) NC+DOX group: 4T1 mouse breast cancer cells were used to perform mammary fat pad tumor formation experiments on wild-type mice. One week later, mice were treated with doxorubicin. The number and dose of doxorubicin were the same as those in group (1). Tumor and cardiac toxicity were evaluated at the same time as in group (1).

[0051] (4) NC group: The mammary fat pad tumor formation experiment was performed on wild-type mice using mouse breast cancer cell line 4T1. Tumor and cardiac toxicity were evaluated at the same time as group (1).

[0052] 2. Experimental results

[0053] (1) Cell experiment results

[0054] Compared with normal breast cells (MCF-10A), circSlc8A1 was down-expressed in breast cancer cells (MCF7). Figure 1 A); Compared with the cardiomyocytes (AC16) not treated with DOX, the expression level of circSlc8A1 in the cardiomyocytes (AC16) treated with DOX was significantly decreased ( Figure 1 B).

[0055] Figure 2 The results are the survival rate and cytotoxicity of cells overexpressing circSlc8A1 in MCF7 and AC16 and then treated with doxorubicin for 24 hours. In the experiment of MCF7 cells, compared with the negative control (OE-NC) group, the survival rate of MCF7 cells overexpressing circSlc8A1 decreased with the increase of doxorubicin concentration and was concentration-dependent, and the cytotoxicity increased with the increase of doxorubicin concentration ( Figure 2 AC); in the experiment of cardiomyocytes AC16, compared with the negative control (OE-NC) group, the cell survival rate of cardiomyocytes AC16 overexpressing circSlc8A1 was improved, and the cardiomyocyte toxicity induced by doxorubicin was significantly reduced ( Figure 2 DF).

[0056] (2) Animal Experiment Results

[0057] The in vivo experiment of doxorubicin treatment used tumor proliferation curves to observe tumor growth. The results showed that circSlc8A1(+ / +) mice had better anti-tumor effects after DOX treatment. Compared with the NC group, the tumor volume of NC group mice was inhibited by 35.0% after doxorubicin treatment (NC+DOX group); the tumor volume of circSlc8A1(+ / +) mice was inhibited by 33.0% after tumor formation (circSlc8A1 group); the tumor volume of circSlc8A1(+ / +) mice was inhibited by 70.0% after doxorubicin treatment (circSlc8A1+DOX group). Figure 3A, B). Compared with breast cancer mice that only overexpressed circSlc8A1 (not treated with doxorubicin) and wild-type breast cancer mice that only treated with doxorubicin (not overexpressing circSlc8A1), the effect of inhibiting tumor volume in breast cancer mice overexpressing circSlc8A1 after doxorubicin treatment was greater than the sum of the effects of the above two, proving that circSlc8A1 and doxorubicin had a synergistic effect in the treatment of breast cancer and achieved unexpected technical effects. At the same time, through mouse ultrasound and cardiac function evaluation, circSlc8A1 improved doxorubicin-induced cardiotoxicity ( Figure 4 A, B).

[0058] In summary, the present invention provides a combined drug for treating breast cancer. The present invention found that circSlc8A1 reduced the survival rate of breast cancer cells treated with doxorubicin and enhanced the chemosensitivity of breast cancer cells, while promoting the cell survival rate of cardiomyocytes induced by doxorubicin chemotherapy. The present invention directly shows through animal experiments that compared with breast cancer mice that only overexpress circSlc8A1 (not treated with doxorubicin) and wild-type breast cancer mice that only treated with doxorubicin (not overexpressing circSlc8A1), the effect of inhibiting tumor volume in breast cancer mice that overexpress circSlc8A1 after doxorubicin treatment is greater than the sum of the effects of the above two, proving that circSlc8A1 and doxorubicin have a synergistic effect in the treatment of breast cancer, achieving unexpected technical effects, and providing a new option for clinical treatment of breast cancer and reduction of cardiotoxicity.

Claims

1. A combined drug, characterized in that: It contains a circular RNA and a chemotherapeutic drug for simultaneous or separate administration, wherein the circular RNA is circSlc8A1.

2. The combined drug according to claim 1, characterized in that: The chemotherapy drug is doxorubicin.

3. The combined drug according to claim 1, characterized in that: It also contains a pharmaceutically acceptable carrier.

4. The combined drug according to claim 1, characterized in that: The combined drug is an injection preparation or an oral preparation.

5. The combined drug according to claim 4, characterized in that: The injection preparation is an intravenous injection preparation, an intraperitoneal injection preparation, an intramuscular injection preparation or a subcutaneous injection preparation; The oral preparation is a decoction, an oral liquid, a granule, a capsule, a powder, a pill or a tablet.

6. Use of the combined drug according to any one of claims 1 to 5 in the preparation of a drug for preventing and / or treating breast cancer.

7. Use of circular RNA in the preparation of a drug for reducing chemotherapy-induced cardiotoxicity, wherein the circular RNA is circSlc8A1.

8. The use according to claim 7, characterized in that The chemotherapy drug is doxorubicin.

9. Use of circular RNA in the preparation of a chemotherapeutic drug sensitizer, wherein the circular RNA is circSlc8A1.

10. The use according to claim 9, characterized in that The chemotherapy drug is doxorubicin.

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