Use of miR408 in preparation of drugs for treating tumors

Abstract

This invention discloses the application of miR408 in the preparation of drugs for treating tumors. The invention found that ginseng miR408 delivered to mice via exosomes maintained good integrity and stability. Overexpression of ginseng miR408 in breast cancer MCF-7 and MDA-MB-231 cells significantly inhibited tumor cell proliferation, migration, and invasion, promoted apoptosis, and downregulated CCND1 mRNA and protein expression levels. Gavage experiments in mice also showed that ginseng miR408 significantly inhibited tumor growth in breast cancer mice. This indicates that ginseng miR408 can enter the body via exosomes to treat breast cancer, providing a new approach to breast cancer treatment, overcoming the adverse reactions of existing drug therapies, and offering a new mechanism of action and research direction for ginseng in breast cancer treatment.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine. Specifically, this invention relates to the application of miR408 in the preparation of drugs for treating tumors. Background Technology

[0002] Breast cancer, one of the most common malignant tumors among women worldwide, seriously threatens women's lives and health. Currently, the main treatments for breast cancer include surgery, chemotherapy, and radiotherapy. Surgery can directly remove tumor tissue, but it is difficult to achieve a complete cure for breast cancer that has metastasized. Chemotherapy can kill cancer cells, but it also damages normal cells, causing a series of serious side effects such as hair loss, nausea, vomiting, and bone marrow suppression, reducing the patient's quality of life. Radiotherapy has a certain effect on local tumor control, but it also has adverse reactions such as radiation dermatitis and radiation pneumonitis.

[0003] MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 19-25 nucleotides in length, playing a crucial role in the transcriptional regulation of gene expression. miRNAs regulate gene expression levels by inhibiting the translation of target gene mRNAs or promoting their degradation; they can also act as anti-cancer miRNAs within tumors by targeting oncogenic mRNAs or tumor-related factors, thus playing an important role. For example, patent CN108096265A discloses miRNA-495's anti-tumor effects, implementation methods, and uses, and describes miR-370-3p inhibiting lung cancer cell growth by inhibiting the lung cancer target MCM2, which can be used to prepare drugs for the prevention or treatment of tumors. Patent CN114645050B discloses an miRNA and its application in the preparation of drugs for treating breast cancer, and also discloses that ach-miR-276a-3 can significantly inhibit breast cancer cell proliferation and tumor growth, providing a new approach for breast cancer treatment.

[0004] Mature miRNAs typically have multiple target genes and can target hundreds of mRNAs. Plant miRNAs almost perfectly complement their target genes. Plant miRNAs play a crucial role in the regulation of biological processes. For example, CN112626112B discloses the application of the maize miR408 gene in regulating plant tolerance to osmotic stress and cultivating plants resistant to osmotic stress; CN113564196A discloses a lettuce Lsa-MIR408 gene and its application in regulating lettuce yield and seed size; CN107574181A discloses miR408 regulating plant photosynthesis and its application; CN116103296A discloses a mulberry miR408 precursor gene and its application in regulating mulberry anthocyanin biosynthesis; and CN105112422B discloses the application of the miR408 gene and UCL in cultivating high-yield rice. However, there are currently no reports on the use of plant-derived miR408 in the treatment of tumors. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of miR408 in the preparation of drugs for treating tumors.

[0006] A second objective of this invention is to provide the use of exosomes containing miR408 in the preparation of medicaments for treating tumors.

[0007] A third objective of this invention is to provide the use of reagents that enhance miR408 expression in the preparation of medicaments for treating tumors.

[0008] A fourth objective of this invention is to provide the application of miR408 in the preparation of CCND1 expression inhibitors.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention employs ultracentrifugation to extract and purify ginseng exosomes, investigating the cross-tissue absorption of ginseng exosomes by mice and cells, and the stability and integrity of miR408 in mouse tissues and cells within exosomes. The role of miR408 in antitumor activity within ginseng exosomes is also analyzed. This invention found that ginseng miRNAs delivered to mice via exosomes maintain good integrity and stability. Overexpression of ginseng miR408 in breast cancer MCF-7 and MDA-MB-231 cells significantly inhibited tumor cell proliferation, migration, and invasion, promoted apoptosis, and downregulated the mRNA and protein expression levels of tumor-related genes. Gavage administration of ginseng miR408 to breast cancer mice significantly inhibited tumor growth. These results indicate that ginseng miR408 can enter the body via exosomes to treat cancer.

[0011] Therefore, the present invention provides the use of miR408 in the preparation of a medicament for treating tumors, wherein the nucleotide sequence of miR408 is 5'-UGCACUGCCUCUUCCCUGGCU-3' (SEQ ID No. 1).

[0012] This invention also provides the application of exosomes containing miR408 in the preparation of drugs for treating tumors, wherein the nucleotide sequence of miR408 is 5'-UGCACUGCCUCUUCCCUGGCU-3'. Exosomes, as nanoscale vesicles secreted by cells, play a crucial role in intercellular communication, capable of carrying various bioactive molecules such as proteins and nucleic acids, crossing biological barriers, and delivering their contents to recipient cells, thereby regulating the function of recipient cells. In the field of tumor therapy, exosomes can serve as natural drug delivery carriers, exerting anti-tumor effects and having wide applications.

[0013] Furthermore, the miR408 is derived from ginseng. Ginseng is a traditional and precious Chinese medicinal herb with various pharmacological activities, including anti-tumor and immune-enhancing effects. Previous studies have found that ginseng contains a variety of miRNAs, and ginseng-derived miRNAs can participate in cross-border regulation between plants and animals through exosome delivery. Ginseng miR408 has potential application value in the treatment of cancer.

[0014] Furthermore, the exosomes are derived from ginseng.

[0015] Furthermore, the method for extracting the exosomes is ultracentrifugation.

[0016] Preferably, the method for extracting exosomes is as follows: collect ginseng tissue fluid, centrifuge at 500g for 10 min at 4°C to remove residual tissue; centrifuge at 2000g for 10 min to remove cells; centrifuge at 10000g for 20 min to remove cell debris; add 1 / 2 volume of Isolation Regent A, centrifuge at 10000g for 5 min, and collect the supernatant; add the same volume of Isolation Regent B, centrifuge at 13500g for 0.5 h, and collect the precipitate.

[0017] The present invention also provides the application of a reagent for enhancing miR408 expression in the preparation of a drug for treating tumors, wherein the nucleotide sequence of miR408 is 5'-UGCACUGCCUCUUCCCUGGCU-3'.

[0018] Furthermore, the drug exerts its effects by inhibiting cell proliferation, migration, and invasion, increasing cell apoptosis, or inhibiting the expression of tumor-related genes.

[0019] Furthermore, the tumor is breast cancer.

[0020] Furthermore, the drug may also include other medically acceptable carriers.

[0021] This invention uses miRBase, miRanda, and targetscan websites to predict the target genes of miR408. GO and KEGG analyses were performed on the prediction results. Among the top 15 signaling pathways with high confidence, CCND1 was involved in 7, indicating that miR408 has a good binding affinity to the 3'UTR of CCND1. After transfecting breast cancer cells with ginseng miR408 mimic, the relative expression levels of CCND1 mRNA and protein were significantly reduced.

[0022] Therefore, the present invention provides the application of miR408 in the preparation of CCND1 expression inhibitors, wherein the nucleotide sequence of miR408 is 5'-UGCACUGCCUCUUCCCUGGCU-3'.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides the application of miR408 in the preparation of drugs for treating tumors. This invention uses ultracentrifugation to extract and purify ginseng exosomes, investigates the cross-tissue absorption of ginseng exosomes by mice and cells, and examines the stability and integrity of miR408 in mouse tissues and cells within the exosomes. The application of miR408 in ginseng exosomes in anti-breast cancer treatment is also analyzed. This invention found that ginseng miR408 delivered to mice via exosomes maintains good integrity and stability. Overexpression of ginseng miR408 in breast cancer MCF-7 and MDA-MB-231 cells significantly inhibited tumor cell proliferation, migration, and invasion, promoted apoptosis, and downregulated CCND1 mRNA and protein expression levels. Gavage administration of ginseng miR408 to breast cancer mice significantly inhibited tumor growth. This indicates that ginseng miR408 can enter the body via exosomes to treat breast cancer, providing a new approach to breast cancer treatment, overcoming the adverse reactions and other problems associated with existing drug treatments, and also providing a new mechanism of action and research direction for ginseng in the treatment of breast cancer. Attached Figure Description

[0025] Figure 1 Transmission electron micrograph of ginseng exosomes. Among them, Figure 1 In the diagram, A and D represent different fields of view for the same sample: A: ruler = 50 nm; B: ruler = 100 nm; C: ruler = 200 nm; D: ruler = 500 nm.

[0026] Figure 2 The A549 cell phagocytosed PKH67 fluorescent exosomes. Figure 2 In the diagram, A and B represent different fields of view of the same sample.

[0027] Figure 3 Colocalization map of ginseng exosomes and intestinal macrophages.

[0028] Figure 4 Colocalization map of ginseng exosomes and hepatic macrophages.

[0029] Figure 5 Colocalization map of ginseng exosomes and splenic macrophages.

[0030] Figure 6 Image from agarose gel electrophoresis. Note: M: Marker; 1: Liver; 2: Small intestine.

[0031] Figure 7 The amplification and melting curves of miR408 are shown.

[0032] Figure 8 GO analysis plot of target prediction for ginseng miR408.

[0033] Figure 9 KEGG analysis plot of target prediction for ginseng miR408.

[0034] Figure 10 CCND1 participated in 7 of the top 15 signal paths with high confidence.

[0035] Figure 11 miR408 exhibits good binding affinity to the 3'UTR of CCND1.

[0036] Figure 12 The effect of ginseng miR408 on the proliferation ability of breast cancer cells.

[0037] Figure 13 The effect of ginseng miR408 on the migration ability of breast cancer cells.

[0038] Figure 14 The effect of ginseng miR408 on the invasive ability of breast cancer cells.

[0039] Figure 15 The effect of ginseng miR408 on the apoptosis ability of breast cancer cells.

[0040] Figure 16 The effect of ginseng miR408 on the predicted target gene CCND1 mRNA in breast cancer cells.

[0041] Figure 17 The effect of ginseng miR408 on the expression of the predicted target gene CCND1 protein in breast cancer cells.

[0042] Figure 18 The growth curve and volume of mouse breast cancer xenografts are shown.

[0043] Figure 19 The tumor is a transplanted tumor in a mouse. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] I. Materials and Methods

[0047] 1. Experimental materials

[0048] All ginseng (Panax ginseng) used in the experiment was purchased from Qingping Traditional Chinese Medicine Market in Guangzhou, Guangdong Province, China, and identified as Panax ginseng CA Mey by Associate Professor Ma Hongyan of Guangdong Pharmaceutical University; DMEM high glucose culture medium and fetal bovine serum were from Gibco, USA; Plant tissue fluid exosome extraction kit S309, BCA protein quantification kit, and PKH26 and PKH67 staining kits were from Shifangjie Company; PBS, trypsin, and penicillin streptomycin were from Hyclone Company; MCF-7 human adenocarcinoma cells were from Cybex; and MDA-MB-231 human adenocarcinoma cells were from Shangen Biotechnology.

[0049] 2. Experimental apparatus

[0050] Refrigerated high-speed centrifuge, Xiangyi H1580R; transmission electron microscope, Hitachi HT7700; NTA particle size analyzer, Malvern NS300; microplate reader, Huaweidelang DR3506; micro-analytical ultrapure water system, Sichuan Woteer Technology Co., Ltd.

[0051] Example 1: Extraction and Validation of Ginseng Exosomes

[0052] I. Experimental Methods

[0053] 1. Ginseng sample pretreatment

[0054] After collecting plant tissue fluid, centrifuge at 500g for 10 min at 4℃ to remove residual plant tissue and collect the supernatant; centrifuge at 2000g for 10 min to remove plant cells and collect the supernatant; centrifuge at 10000g for 20 min to remove plant cell debris and collect the supernatant to extract exosomes or store at 4℃ for later use.

[0055] 2. Exosome extraction

[0056] Transfer the supernatant to a new centrifuge tube and add V (volume ratio). 样 V A Mix Isolation Regent A in a 2:1 ratio; invert and mix 3-5 times, then incubate at 4°C for 5 minutes, followed by centrifugation at 10000g for 5 minutes; transfer the supernatant to a new centrifuge tube, add the same volume of Isolation Regent B; invert and mix 3-5 times, then incubate at 4°C for 1 hour, followed by centrifugation at 13500g for 0.5 hours; discard the supernatant and collect the precipitate, which is the exosome; resuspend the precipitate in 200-500 μL of sterile PBS (or downstream experimental resuspension) according to subsequent experiments, or aliquot and store at -80°C.

[0057] 3. Identification of ginseng exosomes

[0058] (1) Nanoparticle size tracer (NTA) was used to detect the concentration and particle size of ginseng exosomes.

[0059] Take 500 μL of ginseng excretion suspension, dilute it twice with ultrapure water filtered through a 0.22 μm needle filter membrane, and take 1 mL of the diluted liquid for testing on an NTA (model: NS300).

[0060] (2) Transmission scanning electron microscopy (TEM) detection

[0061] Take 10 μL of exosomes, add 10 μL of the sample to a copper grid to precipitate for 1 min, and absorb the floating liquid with filter paper; add 10 μL of uranium acetate to a copper grid to precipitate for 1 min, absorb the floating liquid with filter paper, and dry at room temperature. Perform electron microscopy at 100 kV to obtain transmission electron microscopy imaging results.

[0062] (3) Protein quantification

[0063] Take 20 μL of ginseng exosomes + 20 μL of lysis buffer; prepare standard solutions with concentrations of 0, 0.125, 0.25, 0.5, 0.75, and 1.5 mg / mL protein standards; add 5 μL of each concentration standard to the standard in a 96-well plate (repeat once per well); add 5 μL of sample to the 96-well plate (repeat once per well), add 250 μL of working solution to each well, measure the absorbance using a microplate reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0064] II. Experimental Results

[0065] 1. Results of ginseng exosome transmission scanning electron microscopy (TEM) examination: Figure 1 As shown, exosomes are spherical vesicles, resembling small round saucers, sealed by a lipid double layer, and have a complete morphological structure.

[0066] 2. The particle size analysis results of ginseng exosomes show that the average particle size of ginseng exosomes is 234.8 nm, which meets the exosome particle size standard; its particle concentration is 1.62E+11 Particles / mL, which is relatively high.

[0067] Example 2: Tracer experiment of ginseng exosomes

[0068] I. Experimental Methods

[0069] 1. Co-culture experiment of PKH67-labeled ginseng exosomes with A549 cells

[0070] Preparation of PKH67-labeled ginseng exosome staining solution: The PKH67 cell membrane green fluorescent probe was mixed and diluted with staining buffer; 430 μL of ginseng exosomes were centrifuged at 13500g for 30 min to obtain exosome precipitate, which was resuspended in 430 μL of PKH67 staining working solution. The sample was incubated at 37℃ in the dark for 30 min, and then diluted with 10 times the volume of PBS. Excess dye was removed by centrifugation at 13500g for 30 min. The exosome precipitate was collected and resuspended in 1 mL of PBS to obtain PKH67-labeled exosomes.

[0071] Cell co-culture with labeled exosomes: A549 cells were seeded into 96-well plates, with each well containing 4 × 10⁶ exosomes. 10 Cells / mL. PKH67 fluorescently labeled exosomes were co-cultured with cells for 12 h. Nuclei were stained with DAPI. The original culture medium in the well plate was discarded, and the cells were washed 1-3 times with PBS. DAPI was added to stain the nuclei, completely covering the cells, and incubated for 8-10 min. After washing with PBS, the cells were observed and photographed using an inverted fluorescence microscope (multiple fields of view) to observe the uptake of exosomes by the cells.

[0072] 2. Distribution of PKH26-labeled ginseng exosomes in C57 mice

[0073] GDNPs (ginseng exosomes) were labeled with PKH26 fluorescent marker and administered to C57BL mice by gavage at a volume of 0.8 mL / mouse. The exosome protein concentration was 3.38 mg / mL. Mice were sacrificed at different time points (0 h; 6 h; 12 h) to observe the localization of exosomes in the mouse intestine, liver, and spleen.

[0074] 3. RNA extraction

[0075] Spleens from mice were cryopreserved at -70°C. 1 mL of miRNA extract was added to every 25-50 mg of tissue, and the mixture was ground with liquid nitrogen. The miRNA extract was then added directly to the culture plate to lyse the cells, at a density of 10 cm⁻¹. 2 Add 1 mL of miRNA extractor to the sample and mix thoroughly using a pipette. Extract total RNA and small RNA according to the instructions of the column-based microRNA extraction kit, and then perform electrophoresis on the obtained samples.

[0076] 4. Quantitative expression analysis of miR408

[0077] The target primer sequences were obtained from the Primer Premier 5.0 software design, and the specific sequences are shown in Table 1:

[0078] Table 1 Primer sequences used in the research

[0079]

[0080] cDNA was reverse transcribed using the Maxima Reverse Transcriptase kit, and then subjected to real-time PCR using 2×SG FastqPCR Master Mix (High Rox) on a 7500 real-time PCR instrument. qRT-PCR amplification parameters were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 35 s, for a total of 45 cycles, with GAPDH used as an internal reference gene.

[0081] Table 2 qRT-PCR reaction system (20µL)

[0082]

[0083] 5. Prediction of ginseng miR408 target genes

[0084] The target genes of MIR408 were predicted using the miRBase (http: / / mirbase.org / ), miRanda (http: / / www.microrna.org / microrna / home.do), and targetscan (http: / / www.targetscan.org / ) websites. The prediction results were intersected, and the predicted target genes were imported into the David website for GO and KEGG analysis. The bioinformatics analysis results were visualized using Cytoscape software.

[0085] II. Experimental Results

[0086] 1. For example Figure 2 As shown, after labeling exosomes with PKH67 fluorescent dye, it was found that the exosomes could be phagocytosed by A549 cells.

[0087] 2. For example Figure 3-5 As shown, after mice were gavaged with ginseng exosomes labeled with PKH26 fluorescence, sections of the intestine, liver, and spleen were observed and stained with fluorescence. It was found that ginseng exosomes could co-localize with macrophages (F4 / 80). Macrophages were localized as green fluorescence, while ginseng exosomes were localized as red fluorescence, indicating that they could be taken up by macrophages in the intestine, liver, and spleen.

[0088] 3. Ginseng miRNA quality test results are as follows: Figure 6 As shown, clear miRNA bands were observed at low molecular weight positions on the gel from total RNA of mouse liver and small intestine. These results indicate that miRNA can be extracted from mouse liver and small intestine tissues, and also demonstrate that ginseng miRNA has very high stability, maintaining good integrity even after being delivered to mice via exosomes.

[0089] The results of real-time quantitative PCR detection of ginseng miR408 are as follows: Figure 7 As shown, all amplification curves exhibit clear inflection points, a distinct exponential phase, good parallelism, and a flat baseline with minimal upward movement, indicating excellent amplification. The melting curves of the amplified products all show single, sharp peaks with no other waveforms observed, indicating a homogeneous and uncontaminated product, suggesting the absence of primer dimers and non-specific amplification. This confirms the high stability of ginseng miR408, its ability to enter mice via exosomes, and its incomplete degradation.

[0090] 4. Target prediction of ginseng miR408 is as follows: Figure 8 KEGG analysis results show that the target genes of ginseng miR408 are more involved in the positive regulation of RNA polymerase II promoter transcription in biological processes, and are involved in cell membrane, cytoplasm, and nucleus in cellular components, and in protein binding and DNA binding in molecular functions. KEGG results indicate that ( Figure 9 The ginseng miR408 target gene may be involved in the Wnt signaling pathway, calcium signaling pathway, and MAPK signaling pathway. Among the top 15 signaling pathways with high confidence, CCND1 is involved in 7 of them. Figure 10 ), among which miR408 has good binding affinity to the 3'UTR of CCND1 ( ), Figure 11 ).

[0091] Example 3: Inhibitory effect of ginseng miR408 on breast cancer cells

[0092] I. Experimental Methods

[0093] 1. Cell culture procedures are shown in Table 3.

[0094] Table 3 Cell resuscitation, passage, and cryopreservation procedures

[0095]

[0096] 2. Cell transfection

[0097] (1) Preparation of mimics mother liquor

[0098] After briefly centrifuging the lyophilized powder (3000 rpm, 3 min), dissolve 2ODmiR408 mimic and mimic-NC in 250 μL of DEPC water according to the instructions. Then prepare a 20 μM stock solution, mix it with a pipette, and let it stand at room temperature in the dark for 20 min. Then seal the stock solution device at -20℃ for later use (the synthesis sequences of miR408 mimic and mimic-NC are shown in Table 4).

[0099] Table 4 miRNA synthesis sequences

[0100]

[0101] (2) Transfection

[0102] Using RNA Transfection Reagent (RNATransMate), miRNA408 and mimics-NC mimics were transfected into cells according to the instructions. Cells were collected 24 hours after transfection for subsequent experimental analysis. (RNA Transfection Reagent was provided by Shanghai Sangon Biotech, batch number: E607402-1000)

[0103] 3. Cell proliferation experiment

[0104] Breast cancer cell proliferation was measured using a CCK-8 assay kit. Logarithmic growth phase MCF-7 and MDA-MB-231 cells were digested with trypsin and adjusted to a concentration of 3.75 × 10⁻⁶ cells. 4 / mL and 5×10 3 Cell suspension was aliquoted at a concentration of / mL into sterile, enzyme-free 96-well plates at a volume of 100 μL per well. After culturing for 24 h in a cell culture incubator, the drug was administered. Blank culture medium was added to the control group, the negative control group received the miRNC mimic transfection complex, and the miR408 group received the miR408 mimic transfection complex. After 24 hours of drug treatment, 10% CCK-8 solution was added, and the plates were incubated at 37°C for 30 min in a cell culture incubator. The absorbance at 450 nm was measured using a microplate reader.

[0105] 4. Cell scratch test

[0106] Cell scratch assays are used to assess cell migration ability. Well-grown MCF-7 and MDA-MB-231 cells were seeded at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density in 6-well plates and cultured for 24 h. Three parallel scratches were created in each well using a 200 μL pipette tip. Old culture medium and isolated cells were removed using a pipette, and the wells were washed three times with PBS. The healing process of the scratches was observed and photographed at 0, 24, and 48 hours after drug treatment.

[0107] 5. Transwell invasion experiment

[0108] Transwell assays were used to measure cell invasion ability. Matrigel matrix was diluted 1:8 with basal medium, thoroughly mixed, and added to the upper chamber (100 μL). The mixture was then incubated at 37°C for 1 h to solidify into a membrane. Starved MDA-MB-231 cells (5 × 10⁶ cells / year) were then... 4Cells were added to the upper chamber along with basal medium containing the transfection complex, and 500 μL of DMEM containing 15% FBS was added to the lower chamber. The 24-well plate was incubated for 24 h. After removing the chambers, the cells were fixed with 500 μL of 4% paraformaldehyde for 20 min and stained with 500 μL of crystal violet staining solution for 10 min. The cells were washed twice with PBS and observed under an inverted fluorescence microscope, and the images were recorded.

[0109] 6. Apoptosis experiment

[0110] Flow cytometry was used to detect apoptosis. Well-grown MCF-7 and MDA-MB-231 cells were seeded into 6-well plates and incubated for 24 h. Cells were then treated with basal medium containing the miRNA mimic transfection complex and incubated for another 24 h. After centrifugation at 1000 rpm for 5 min, cells were collected, washed twice with PBS, and resuspended in 1× binding buffer (1×10⁻⁶). 6 Cells were added to a solution of 5 μL Annexin V-FITC and 5 μL PI (propidium iodide) and incubated in the dark at room temperature for 15 min before being analyzed by flow cytometry.

[0111] 7. Effects of ginseng miR408 on the expression levels of breast cancer-related genes

[0112] (1) Cell RNA extraction: The RNA extraction procedure is shown in Table 5.

[0113] Table 5. Cell RNA Extraction Procedure

[0114]

[0115] (2) Real-time quantitative PCR

[0116] Prepare the following mixture in an RNase-free octet using a reverse transcription kit (MightyScript First-Strand cDNA Synthesis Master Mix) and gently pipette to mix. Follow the reverse transcription kit instructions; Table 6 shows the reverse transcription reaction system, which should be prepared on ice. The reaction conditions are: 25°C for 5 minutes, 55°C for 15 minutes, 85°C for 5 minutes, one cycle, then store on ice at -20°C for short-term storage.

[0117] Table 6. Reverse transcription reaction preparation system (20 μL)

[0118]

[0119] Primer sequences were designed using Oligo 7 software. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (primer information is shown in Table 7). The reaction system was configured according to Table 8, and the reaction conditions were: pre-denaturation at 95℃ for 3 min, followed by denaturation at 95℃ for 5 s, and annealing extension at 60℃ for 20 s, for a total of 40 cycles. The CT values ​​were calculated and analyzed.

[0120] Table 7 Primer Sequences

[0121]

[0122] Table 8 Reaction system (20 μL)

[0123]

[0124] 8. Effects of ginseng miR408 on the expression levels of breast cancer-related proteins

[0125] (1) Extraction of cell proteins

[0126] ① Line the cells in a 6-well plate. After the cells adhere, change the medium and administer the drug. When the cell confluence reaches 80% or more, extract the protein. Take out the 6-well plates transfected with miRNA mimics for 24h and 48h, remove the culture medium, and wash the cells twice with 1mL PBS.

[0127] ② Add 100× protease inhibitor and 100× phosphatase inhibitor to the lysis buffer at a ratio of 1:100. Add 200 μL of the prepared lysis buffer to each well, shake the plate, and place it on ice for 3-5 min. Scrape off cells with a cell scraper, transfer them to a 1.5 mL sterile centrifuge tube, place on ice for 20 min with repeated shaking, centrifuge at 12000 rpm and 4℃ for 5 min, collect the supernatant to a new centrifuge tube and record the volume.

[0128] ③ To ensure that the protein concentration of each group is maintained, 5 μL of supernatant is taken and the cell protein concentration is measured using the BCA kit. The protein concentration of each group is calculated and adjusted to be consistent.

[0129] ④ Add protein loading buffer to the supernatant with adjusted protein concentration, dilute and vortex to mix, place in boiling water at 100℃ for 3-5 minutes, let cool naturally and then freeze at -20℃.

[0130] (2) Western blot

[0131] ① Gel preparation: Assemble the gel preparation equipment and prepare the separating gel and stacking gel using the PAGE Gel Rapid Preparation Kit (10%). First, pour the separating gel, add ethanol and press it flat, and let it stand at room temperature for 15 minutes; pour off the ethanol, blot dry, add the stacking gel, insert a comb, wipe off the excess gel, and after the gel solidifies, put it into the electrophoresis tank and pour in the electrophoresis buffer.

[0132] ② Sample loading: Melt the above protein sample at room temperature, vortex mix, remove the comb in the electrophoresis buffer, add 10 μL of WB protein sample to each well, and add 2 μL and 4 μL of protein marker to the leftmost and rightmost wells, respectively.

[0133] ③ Electrophoresis: Add electrophoresis buffer, stack the gel at 80V for 10 minutes, and when the sample reaches the separating gel, change the voltage to 120V for 1 hour of electrophoresis.

[0134] ④ Transfer: Preparing transfer materials in the later stages of electrophoresis: Pour 4℃ transfer buffer into a medical tray. Immerse the PVDF membrane in methanol for 30 seconds to activate it, then place it in the transfer buffer. After electrophoresis, cut off the target protein region of the stacking gel in the medical tray. Assemble the transfer clips in the following order (blackboard, sponge, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, sponge, whiteboard). After assembly, remove any air bubbles and place the tray in a transfer tank on an ice-water bath. Transfer at 300mA for 1 hour.

[0135] ⑤ Blocking: Cut the PVDF membrane region containing the target protein according to the protein marker, place it in a WB-specific incubation box, wash 3 times with TBST solution (10 min / wash), add WB-specific rapid blocking solution and slowly block on a shaker for 10 min.

[0136] ⑥ Primary antibody: After recovering the blocking solution, wash the membrane three times with TBST solution (10 min / time), add the primary antibody and incubate overnight at 4℃; Secondary antibody: The next day, recover the primary antibody, wash the PVDF membrane three times with TBST solution (10 min / time), add the secondary antibody and incubate at low speed on a shaker at room temperature for 2 hours, recover the secondary antibody and wash the membrane three times with TBST solution (10 min / time).

[0137] ⑦ Development: Add freshly prepared developer to the darkroom, immerse the PVDF membrane and incubate in the dark for 60 seconds, develop and photograph it in a chemiluminescence analyzer, and quantitatively analyze the developed bands using Image J.

[0138] 9. Data Analysis

[0139] The experimental data were analyzed using GraphPad Prism 9.5 statistical software. All data are expressed as mean ± standard error (±s). One-way ANOVA was used for statistical analysis between groups. A p-value < 0.05 was considered statistically significant.

[0140] II. Experimental Results

[0141] 1. Results of the inhibition of MCF-7 and MDA-MB-231 proliferation by ginseng miR408 are as follows: Figure 12As shown, compared with the control group, the NC group showed no significant difference in cell proliferation (P>0.05), while the miR408 group significantly inhibited the proliferation of MCF-7 and MDA-MB-231 cells (P<0.001; P<0.001). This indicates that overexpression of ginseng miR408 can inhibit the proliferation of MCF-7 and MDA-MB-231 cells.

[0142] 2. Results of ginseng miR408 inhibiting the migration ability of MCF-7 and MDA-MB-231 are as follows: Figure 13 As shown, 24 h and 48 h after transfecting MCF-7 and MDA-MB-231 cells with ginseng miR408 mimic, there was no significant difference in scratch healing area between the NC group and the control group (P>0.05), while the miR408 group significantly reduced the scratch healing area of ​​MCF-7 and MDA-MB-231 cells (P<0.001; P<0.001). This indicates that ginseng miR408 can significantly inhibit the migration ability of breast cancer cells.

[0143] 3. Results of the ability of ginseng miR408 to inhibit the invasion of MDA-MB-231 are as follows: Figure 14 As shown, both the control group and the NC group contained a large number of invasive cells. Compared with the control group, the miR408 group significantly reduced the number of invasive cells (P<0.001). This indicates that ginseng miR408 has a significant effect on inhibiting the invasive ability of breast cancer cells MDA-MB-231.

[0144] 4. Results of ginseng miR408 promoting apoptosis in MCF-7 and MDA-MB-231 cells are as follows: Figure 15 As shown, in MCF-7 and MDA-MB-231 cells, compared with the control and NC groups, transfection with ginseng miR408 mimic increased the proportion of apoptotic cells (P<0.001; P<0.001). This indicates that ginseng miR408 can promote apoptosis in MCF-7 and MDA-MB-231 cells.

[0145] 5. Effects of ginseng miR408 on the expression of tumor-related gene CCND1 mRNA and protein, such as... Figure 16-17As shown, 24 h after transfecting MCF-7 and MDA-MB-231 cells with ginseng miR408 mimic, the relative expression level of CCND1 mRNA in the miR408 group was significantly lower in both cell types compared with the control and NC groups (P<0.001; P<0.001), with a more pronounced effect in MCF-7 cells. 48 h after transfecting MCF-7 and MDA-MB-231 cells with ginseng miR408 mimic, the relative expression level of CCND1 protein in the miR408 group was significantly lower in both cell types compared with the control and NC groups (P<0.001; P<0.01).

[0146] Example 4: Inhibitory effect of ginseng miR408 on breast cancer mice

[0147] I. Experimental Methods

[0148] 1. Establish a mouse model of breast cancer xenograft

[0149] 4T1 tumor cell lines in the logarithmic proliferation phase were selected, and the cell pellet was collected by centrifugation after trypsin digestion. The cell density was adjusted to 1×10⁶ cells / year using basal culture medium. 6 Single-cell suspensions were prepared by repeated pipetting and aspiration using a pipette. The homogenized suspension was aliquoted into sterile centrifuge tubes and stored at 4°C for a short period. Before cell seeding, the cell suspension was brought to room temperature and gently mixed. 100 μL of the suspension was subcutaneously injected into the left pectoral fourth mammary fat pad region of the experimental group mice. Model validation was performed 7 days after injection; successful tumor model establishment was defined as the observation of significant solid tumor growth at the injection site.

[0150] 2. Experimental grouping and drug administration

[0151] Forty-eight SPF-grade female BALB / c mice, sourced from Guangdong Zhiyuan Biotechnology Co., Ltd., were housed in the SPF-grade animal room of the Experimental Animal Center of Guangdong Pharmaceutical University (Ethics Code: gdpulacspf2022602). The room temperature was maintained at approximately 25℃, and the humidity at 40%-70%. The mice were acclimatized for one week before the experiment, with a 12-hour day-night cycle and free access to water and food. After tumor modeling, the mice were randomly divided into four groups: a model control group (C group), a low-dose ginseng miR408 group (L group), a medium-dose ginseng miR408 group (M group), a high-dose ginseng miR408 group (H group), a ginseng miR-NC group (N group), and a cisplatin group (P group), with eight mice in each group. Group C: PBS 0.1 mL / day by gavage; Ginseng miR408 administration groups: Group L 150 pM / day by gavage, Group M 300 pM / day by gavage, Group H 600 pM / day by gavage, with a gavage volume of 0.1 mL / day, for 21 consecutive days. Group P: Cisplatin 3 mg / kg intraperitoneally, 3 times / day.

[0152] 3. Determination of tumor inhibition rate in mice

[0153] Seven days after inoculation, the long diameter (a) and short diameter (b) of the transplanted tumor were measured every three days using electronic digital calipers. The volume was calculated using the formula V = 0.5 × a × b. 2 Twenty-one days after drug administration, the tumor was euthanized via cervical dislocation according to ethical guidelines for laboratory animals. The tumor tissue was completely removed, photographed, and weighed. The tumor inhibition rate of the drug was calculated using the following formula:

[0154] Tumor inhibition rate (%) = [1 - (mean tumor weight in the treatment group / mean tumor weight in the model group)] × 100%.

[0155] II. Experimental Results

[0156] 1. Growth curve and volume of mouse breast cancer xenografts

[0157] The results of ginseng miR408 inhibiting the growth of breast cancer xenografts in mice are as follows: Figure 18 As shown, the results indicated that the tumor volume in group C increased rapidly, reaching a significant increase by day 21. Group N showed a similar trend to group C, indicating rapid tumor growth in both the absence of intervention and miR-NC intervention. During the experiment, the tumor volume growth rates in groups L, M, and H were significantly lower than those in group C, with group M showing the most significant inhibitory effect. This difference became increasingly significant over time, indicating that each treatment group had an inhibitory effect on the growth of mouse breast cancer xenografts.

[0158] 2. Tumor inhibition rate of mice in each group

[0159] Tumor samples were removed from the mice after gavage, and the results were as follows: Figure 19 As shown in Table 9, the size of the tumor can be clearly observed. Compared with group C, the tumor in group M is significantly smaller. The tumor inhibition rates of each group of mice are shown in Table 9: Compared with group C, the tumor weight of groups L, M, H, and P is significantly reduced (P<0.05). The tumor inhibition rates of groups L, M, and H are 24.78%, 54.44%, and 29.62%, respectively. This indicates that group M has the strongest tumor inhibition effect, while groups L and H also have a certain inhibitory effect on tumor inhibition.

[0160] Table 9 Tumor inhibition rate of mice in each group

[0161]

Claims

1. The application of miR408 as the sole active ingredient in the preparation of a drug for treating breast cancer, characterized in that, The nucleotide sequence of miR408 is 5'-UGCACUGCCUCUUCCCUGGCU-3'.

2. The application of an exosome containing miR408 as the sole active ingredient in the preparation of a medicament for treating breast cancer, characterized in that, The nucleotide sequence of miR408 is 5'-UGCACUGCCUCUUCCCUGGCU-3'.

3. The application according to claim 1 or 2, characterized in that, The miR408 is derived from ginseng.

4. The application according to claim 2, characterized in that, The exosomes are derived from ginseng.

5. The application according to claim 2, characterized in that, The exosomes were extracted using ultracentrifugation.

6. The application according to claim 1 or 2, characterized in that, The drug works by inhibiting tumor cell proliferation, migration, and invasion, increasing tumor cell apoptosis, or inhibiting the expression of the tumor-related gene CCND1.

7. The application according to claim 1 or 2, characterized in that, The drug may also include other medically acceptable carriers.

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