Application of tea polyphenol in prevention and treatment of triple negative breast cancer

Abstract

The invention relates to the technical field of oncology and drug development, in particular to application of tea polyphenol in prevention and treatment of triple-negative breast cancer, and particularly discloses application of tea polyphenol in any one or more of the following scenes: a1) inhibiting proliferation of triple-negative breast cancer cells or preparing a product for inhibiting proliferation of triple-negative breast cancer cells, a2) inhibiting proliferation of triple-negative breast cancer cells, a3) inhibiting proliferation of triple-negative breast cancer cells, a4) inhibiting proliferation of triple-negative breast cancer cells and a4) inhibiting proliferation of triple-negative breast cancer cells; a2) inhibiting migration and invasion of triple negative breast cancer cells or preparing a product for inhibiting migration and invasion of triple negative breast cancer cells; a3) inhibiting the survival of the triple-negative breast cancer or preparing a product for inhibiting the survival of the triple-negative breast cancer; a4) preparing a product for preventing and / or treating triple negative breast cancer; and a5) detecting and monitoring the regulation effect of the tea polyphenol on the miRNA expression of the triple negative breast cancer cells. The tea polyphenol inhibits unlimited rapid proliferation, invasion and migration of triple negative breast cancer cells by influencing the cell cycle process, inducing cell apoptosis, interfering cytoskeleton recombination and inhibiting cell adhesion, and improves the prognosis and survival rate of triple negative breast cancer patients.

Description

Technical Field

[0001] The present application relates to the technical field of oncology and drug development, and more specifically, it relates to the application of tea polyphenols in preventing and treating triple-negative breast cancer. Background Art

[0002] Triple-Negative Breast Cancer (TNBC) is a subtype of breast cancer characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC is highly malignant, usually presents at a young age, and is characterized by high invasiveness, high metastasis rate, rapid growth, and poor prognosis. Due to the lack of specific targets, hormone therapy and targeted therapy are ineffective, while conventional chemotherapy and radiotherapy lack selectivity and specificity, and have serious toxic side effects and tolerability issues. Therefore, the development of new treatment strategies is crucial to improving the prognosis of TNBC patients.

[0003] Tea polyphenols are natural compounds extracted from tea leaves, especially green tea, and are mainly composed of polyphenolic compounds such as flavanols (catechins), flavonoids, anthocyanins and phenolic acids. Among them, flavanols (catechins) are the main ones, accounting for 60-80% of the total tea polyphenols. Catechins are mainly epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), with EGCG having the highest content. Tea polyphenols have multiple biological functions such as antioxidant, anti-tumor, anti-inflammatory and antibacterial, anti-radiation, anti-stroke, lowering blood lipids and delaying aging, and have been widely used in food health care, clinical medicine and beauty cosmetics. However, the comprehensive effect of tea polyphenols on the prevention and treatment of triple-negative breast cancer, especially the comprehensive effect of tea polyphenols on the expression of small molecule RNA microRNA (miRNA) related to triple-negative breast cancer and its application need further study. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an application of the natural compound tea polyphenols in the prevention and treatment of triple-negative breast cancer, effectively inhibit the survival, proliferation and migration of triple-negative breast cancer cells, and combine the regulatory effect of tea polyphenols on the expression of miR-21 and miR-10b, in order to provide new treatment strategies and monitoring methods for the treatment of triple-negative breast cancer.

[0005] The application of tea polyphenols in the prevention and treatment of triple-negative breast cancer provided in this application adopts the following technical solution: The application of tea polyphenols in the prevention and treatment of triple-negative breast cancer is applied in any one or more of the following scenarios: a1) Inhibiting the proliferation of triple-negative breast cancer cells or preparing products that inhibit the proliferation of triple-negative breast cancer cells; a2) Inhibiting the migration and invasion of triple-negative breast cancer cells or preparing products that inhibit the migration and invasion of triple-negative breast cancer cells; a3) inhibiting the survival of triple-negative breast cancer or preparing products that inhibit the survival of triple-negative breast cancer; a4) preparing products for preventing and / or treating triple-negative breast cancer; a5) Detect and monitor the regulatory effect of tea polyphenols on miRNA expression in triple-negative breast cancer cells.

[0006] Preferably, the triple-negative breast cancer cells are MDA-MB-231.

[0007] Preferably, the concentration of the tea polyphenols is 1 / 2IC50 concentration - 2IC50 concentration, that is, 67.85 μg / mL - 271.4 μg / mL.

[0008] Preferably, the IC50 concentration is 135.7 μg / mL.

[0009] Preferably, the miRNA is miR-21 and miR-10b.

[0010] In summary, the present application has at least the following beneficial effects: 1. The present invention discloses the changes in the survival and proliferation rate of MDA-MB-231 cells after treatment with tea polyphenols at different concentrations. The survival and proliferation rate of cells in the tea polyphenols 100 μg / mL treatment group was only 59.84% of that in the control group, and the survival and proliferation rate of cells in the tea polyphenols 300 μg / mL treatment group was only 26.09% of that in the control group. The half inhibitory concentration (IC50) of tea polyphenols on the survival and proliferation of MDA-MB-231 cells was 135.7 μg / mL.

[0011] 2. Within the concentration range of this application, tea polyphenols, 1 / 2IC50 concentration - 2IC50 concentration, i.e. 67.85 μg / mL - 271.4 μg / mL, can significantly inhibit the survival and proliferation of MDA-MB-231 cells. The mechanism may be to achieve effective inhibition of the survival and proliferation of MDA-MB-231 cells by affecting the cell cycle process and inducing cell apoptosis.

[0012] 3. Tea polyphenols can significantly inhibit the migration of MDA-MB-231 cells within the concentration range of this application. The differences in the results at different concentrations are statistically significant. The mechanism may be that tea polyphenols significantly inhibit the migration ability of MDA-MB-231 cells by interfering with cytoskeleton reorganization and inhibiting cell adhesion.

[0013] 4. The present invention also provides two miRNA molecules regulated by tea polyphenols in a triple-negative breast cancer cell line (MDA-MB-231), namely miR-21 and miR-10b; the expression of miR-21 and miR-10b in the cancer cells of the tea polyphenol 1 / 2 IC50 concentration treatment group was downregulated by 96.4% and 98.4% respectively compared with the control group, and the difference was very significant; this inhibitory regulatory effect resulted in the blocking of the signal pathways and biological effects related to miR-21 and miR-10b, thereby inhibiting the malignant behavior of MDA-MB-231 cells including unlimited rapid proliferation, invasion and metastasis; detecting the expression levels of miR-21 and miR-10b can be used as an indicator for judging the malignancy, treatment effect and prognosis of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the changes in the survival and proliferation rate of MDA-MB-231 cells after treatment with different concentrations of tea polyphenols; Figure 2 It is a schematic diagram of the changes in the migration rate of MDA-MB-231 cells after treatment with different concentrations of tea polyphenols; Figure 3 is a schematic diagram of the changes in miR-21 expression after treatment with different concentrations of tea polyphenols; Figure 4 This is a schematic diagram showing the changes in miR-10b expression after treatment with different concentrations of tea polyphenols. DETAILED DESCRIPTION

[0015] The present application is further described in detail with reference to the following examples. Example

[0016] Example 1 Using CCK-8 to detect the effects of different concentrations of tea polyphenols on cancer cell survival and proliferation Press 1*10 4Density of cells / well MDA-MB-231 cells were seeded in a 96-well plate and cultured in an incubator (37°C, 5% CO2) for 24 hours until the cells adhered to the wall and grew to a cell density of about 70%. Cells were treated with different concentrations of tea polyphenols (0μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 300μg / mL, 400μg / mL, 500μg / mL, 600μg / mL) for 24 hours. CCK-8 working solution was prepared at a ratio of CCK-8:DMEM=1:9, all the culture medium in each well was discarded, CCK-8 working solution was added to each well, and incubated in an incubator for 2 hours. When the color of the culture medium in the control group changed from light red to orange, the OD value was read at 450nm using an enzyme reader. Compare the changes in cell viability and number in different treatment groups, relative viability and number % = (OD value of experimental well / OD value of control well) X 100%.

[0017] The results are as follows Figure 1 As shown, tea polyphenols treatment had a significant inhibitory effect on the survival and proliferation rate of MDA-MB-231 cells. The survival and proliferation rate of cells in the tea polyphenols 100μg / mL treatment group was only 59.84% of that in the control group, and the survival and proliferation rate of cells in the tea polyphenols 300μg / mL treatment group was only 26.09% of that in the control group. The half inhibitory concentration (IC50) of tea polyphenols on MDA-MB-231 cells was 135.7μg / mL.

[0018] Example 2 Using Transwell to detect the effects of different concentrations of tea polyphenols on cancer cell migration The experiment was divided into a control group and a treatment group with different concentrations of tea polyphenols. Treatment group 1 was a 1 / 2IC50 group, treatment group 2 was an IC50 group, and treatment group 3 was a 2IC50 group. Equal amounts of MDA-MB-231 cells were inoculated in the Transwell chamber, and DMEM medium without fetal bovine serum (FBS) was added to the upper layer, and medium containing 20% ​​FBS was added to the lower layer. After 48 hours of culture, the cells were fixed with 4% paraformaldehyde, the untransferred cells on the upper layer of the Transwell chamber membrane were scraped off, and the transferred cells on the lower layer were stained with 0.2% crystal violet to make them visible, and the number of cells that migrated to the lower chamber membrane was counted.

[0019] The results are as follows Figure 2 As shown. Figure 2 A shows that the cells in the control group migrated more and more densely, while the cells in the 1 / 2IC50, IC50 and 2IC50 groups treated with different concentrations of tea polyphenols migrated slowly, and the number of cells migrating across the chamber membrane was significantly reduced. Figure 2B shows that the number of cancer cell migration in the tea polyphenol 1 / 2 IC50 concentration (67.85 μg / mL) treatment group was 87.85% lower than that in the control group. The number of cancer cell migration in the tea polyphenol IC50 concentration (135.7 μg / mL) and 2 IC50 concentration (271.4 μg / mL) treatment groups was 89.81% and 94.65% lower than that in the control group, respectively, and the difference was statistically significant.

[0020] Example 3 Detection of the effect of tea polyphenols on the expression of miR-21 and miR-10b in cancer cells using RT-qPCR MDA-MB-231 cells were treated with tea polyphenols, and the expression changes of miR-21 and miR-10b were detected by RT-qPCR. The details are as follows: 1. RNA extraction: Centrifuge the collected cells at 4°C and 3000 rpm for 3 minutes, discard the supernatant, add 500μL FreeZol Reagent, vortex until the solution is fully mixed, and store at -80°C. Take out the sample and dissolve it on ice, add 150μL Dilution Buffer to the lysate, vortex until the solution is fully mixed, and let it stand at room temperature for 10 minutes. Centrifuge at 12000rpm for 15 minutes at room temperature, carefully pipette the supernatant (about 500μL) into a new centrifuge tube, add an equal volume of isopropanol, mix thoroughly by inverting, let it stand at room temperature for 15 minutes, and centrifuge at 12000 rpm for 15 minutes at room temperature. Discard the supernatant, add 1 mL 75% ethanol, flick the bottom of the tube lightly to suspend the precipitate, and invert it several times, centrifuge at 8000 rpm for 5 minutes at room temperature, discard the supernatant, let it dry at room temperature, and add 20μL DEPC water to dissolve the precipitate.

[0021] 2. cDNA Synthesis Stem-loop primer method: To detect the expression of miRNA using the stem-loop primer method, it is necessary to involve specific stem-loop primers and PCR primers for the specific sequence of miRNA. During the PCR amplification process, SYBR-green color development is used to detect the amplification of the target miRNA in real time and quantitatively. The specific information of the stem-loop primer and the corresponding PCR amplification primer is as follows: miR-21 stem-loop primers: 5'-GCGCGTGAGCAGGCTGGAGAAATTAACCACGCGCGGAACG-3' (SEQ ID NO: 1) miR-10b stem-loop primers: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACAAATT-3' (SEQ ID NO: 2) miR-21 PCR forward primer: 5'-CGCAAGGATGACACG-3' (SEQ ID NO: 3) miR-21 PCR reverse primer: 5'-GAGCAGGCTGGAGAA-3' (SEQ ID NO: 4) miR-10b PCR forward primer: 5'-ACACTCCAGCTGGGTACCCTGTCGAACCGAA-3' (SEQ ID NO: 5) miR-10b PCR reverse primer: 5'-TGGTGTCGTGGAGTCG-3' (SEQ ID NO: 6) Reverse transcription reaction substrate: 0.5 μL M-MLV (H-) reverse transcriptase, 0.5 μL RNase inhibitor, 2 μL 5× RT buffer, 0.5 μL dNTP mixture, 1 μL (500 ng) RNA and 5 μL DEPC water.

[0022] Reaction conditions: pre-denaturation at 50°C for 45 min, denaturation at 85°C for 5 min.

[0023] 3. Perform fluorescent quantitative PCR on cDNA In a new 0.2 mL PCR reaction tube, add 15 µL ChamQ SYBR qPCR Master Mix, 0.8 µL each of PCR forward and reverse primers (10 µM), 1.8 µL template cDNA, and 14 µL deionized water.

[0024] The reaction conditions were: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for a total of 40 cycles.

[0025] 4. Data processing Fluorescence quantitative PCR quantitative detection of the obtained miRNA C T Value, compare the changes in the expression of cancer-promoting miR-21 and miR-10b in cells treated with tea polyphenols, relative expression = 2^ -△△Ct .

[0026] The results are as follows Figure 3 and Figure 4 shown. Figure 3The results show the changes in miR-21 expression in the tea polyphenols treatment groups with different concentrations. The results show that the expression of miR-21 in cancer cells in the tea polyphenols 1 / 2IC50 concentration (67.85μg / mL) treatment group decreased significantly, and the inhibitory effect reached 96.4%. The expression of miR-21 in cancer cells in the tea polyphenols IC50 concentration (135.7μg / mL) and 2IC50 concentration (271.4μg / mL) treatment groups was inhibited by 98.3%.

[0027] Figure 4 The results show the changes in miR-10b expression in the tea polyphenols treatment groups with different concentrations. The results show that the expression of miR-10b in cancer cells in the tea polyphenols 1 / 2IC50 concentration (67.85μg / mL) treatment group decreased significantly, and the inhibitory effect reached 96.36%. The expression of miR-10b in cancer cells in the tea polyphenols IC50 concentration (135.7μg / mL) and 2IC50 concentration (271.4μg / mL) treatment groups was also inhibited to a similar extent.

[0028] Studies have shown that tea polyphenols can inhibit the proliferation and migration of cancer cells and induce apoptosis of cancer cells through various mechanisms. miRNA is a class of small non-coding RNA molecules that are involved in regulating gene expression and cell function. miRNA has been identified as a key regulator of complex biological processes related to normal physiology and the pathogenesis of diseases (such as cancer, muscle disease, cardiovascular disease, autoimmune disease and neurodegeneration). Abnormal expression of miRNA is caused by gene mutations in various cancer types and plays an important role in the occurrence, development and metastasis of cancer through dysregulation of target gene expression.

[0029] miR-21 and miR-10b are two cancer-related miRNAs that are highly expressed in triple-negative breast cancer and are associated with the proliferation and migration of breast cancer cells. Overexpression of miR-21 and miR-10b promotes the invasiveness of cancer cells and is associated with a poor prognosis.

[0030] Tea polyphenols can significantly inhibit the survival and proliferation of MDA-MB-231 cells within a certain concentration range. The cell survival and proliferation rates of the tea polyphenols-treated group were significantly reduced. The cell survival and proliferation rates of the tea polyphenols 100μg / mL-treated group were only 59.84% of the control group, and the cell survival and proliferation rates of the tea polyphenols 300μg / mL-treated group were only 26.09% of the control group. The half inhibitory concentration (IC50) of tea polyphenols on the survival and proliferation of MDA-MB-231 cells was 135.7μg / mL. Tea polyphenols effectively inhibited the survival and proliferation of MDA-MB-231 cells by affecting the cell cycle process and inducing cell apoptosis.

[0031] Tea polyphenols can significantly inhibit the migration of MDA-MB-231 cells within a certain concentration range. The number of cancer cell migration in the tea polyphenol 1 / 2IC50 concentration (67.85μg / mL) treatment group was 87.85% lower than that in the control group. The number of cancer cell migration in the tea polyphenol IC50 concentration (135.7μg / mL) and 2IC50 concentration (271.4μg / mL) treatment groups was 89.81% and 94.65% lower than that in the control group, respectively, and the difference was statistically significant. Tea polyphenols significantly inhibited the migration ability of MDA-MB-231 cells by interfering with cytoskeleton reorganization and inhibiting cell adhesion.

[0032] The present invention also provides two miRNA molecules regulated by tea polyphenols in a triple-negative breast cancer cell line (MDA-MB-231), namely miR-21 and miR-10b. The expression of miR-21 and miR-10b in the cancer cell group treated with tea polyphenols at a concentration of 1 / 2 IC50 was downregulated by 96.4% and 98.4% respectively compared with the control group, and the difference was very significant. The treatment group with a higher concentration of tea polyphenols showed a similar inhibitory effect. This inhibitory regulatory effect may lead to the blocking of signal pathways and biological effects related to miR-21 and miR-10b, thereby inhibiting the malignant behavior of MDA-MB-231 cells, including unlimited rapid proliferation, invasion and metastasis. Detecting the expression levels of miR-21 and miR-10b can be used as an indicator for judging the malignancy and therapeutic effect of triple-negative breast cancer.

[0033] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. The application of tea polyphenols in preventing and treating triple-negative breast cancer, characterized in that: Apply in one or more of the following scenarios: a1) Inhibiting the proliferation of triple-negative breast cancer cells or preparing products that inhibit the proliferation of triple-negative breast cancer cells; a2) Inhibiting the migration and invasion of triple-negative breast cancer cells or preparing products that inhibit the migration and invasion of triple-negative breast cancer cells; a3) inhibiting the survival of triple-negative breast cancer or preparing products that inhibit the survival of triple-negative breast cancer; a4) preparing products for preventing and / or treating triple-negative breast cancer; a5) Detect and monitor the regulatory effect of tea polyphenols on miRNA expression in triple-negative breast cancer cells.

2. The use of tea polyphenols in preventing and treating triple-negative breast cancer according to claim 1, characterized in that: The triple-negative breast cancer cell line is MDA-MB-231.

3. The use of tea polyphenols in preventing and treating triple-negative breast cancer according to claim 1, characterized in that: The concentration of the tea polyphenols is 1 / 2IC50 concentration-2IC50 concentration, that is, 67.85 μg / mL-271.4 μg / mL.

4. The use of tea polyphenols in preventing and treating triple-negative breast cancer according to claim 3, characterized in that: The IC50 concentration of the tea polyphenols is 135.7 μg / mL.

5. The use of tea polyphenols in preventing and treating triple-negative breast cancer according to claim 1, characterized in that: The miRNAs are miR-21 and miR-10b.

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