Application of MEX3D as a detection marker and therapeutic target for triple-negative breast cancer

Abstract

The present invention relates to the application of MEX3D as a detection marker and therapeutic target for triple-negative breast cancer, and belongs to the field of biomedical technology. The present invention establishes the RNA binding protein spectrum of the triple-negative breast cancer cell line MDA-MB-231 and the estrogen receptor-positive breast cancer cell line MCF-7 based on the mRNA interaction capture method, and performs bioinformatics analysis to screen out the RNA binding protein MEX3D that affects the proliferation and invasion of triple-negative breast cancer cells. MEX3D can be used as a detection marker and therapeutic target for triple-negative breast cancer, which is expected to solve the problem of the current lack of effective diagnosis and treatment methods for triple-negative breast cancer, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of MEX3D as a detection marker and treatment target for triple-negative breast cancer. Background Art

[0002] Breast cancer is one of the leading causes of cancer death in women. It is also a common malignant tumor in women, and its incidence is on the rise. The diagnosis and classification of breast cancer have always relied on the analysis of tumor morphology and the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Breast cancer lacking these three proteins is called triple-negative breast cancer (TNBC). Triple-negative breast cancer accounts for about 15-20% of breast cancer. Compared with other types of breast cancer, triple-negative breast cancer has stronger aggressiveness, younger age of onset, strong metastasis, poor prognosis, high recurrence, and low survival rate. The molecular mechanism of recurrence of triple-negative breast cancer has not been elaborated in detail. Therefore, until now, there is no good targeted treatment for triple-negative breast cancer patients. Although patients with early detection of triple-negative breast cancer can be cured by chemotherapy, for those patients with metastasis, the average survival time is only 13-18 months with current treatment methods.

[0003] Triple-negative breast cancer is a difficult disease to treat clinically due to its high heterogeneity and lack of suitable therapeutic targets. Chemotherapy is currently the main treatment for triple-negative breast cancer, including anthracyclines, taxanes, and cisplatin, which mainly destroy the function of tumor cells. Because a large number of people do not improve after chemotherapy, whether different chemotherapy methods should be selected according to the subtype of triple-negative breast cancer has caused great controversy.

[0004] Although triple-negative breast cancer accounts for a relatively small proportion of breast cancer, it has a disproportionate mortality rate among breast cancer patients. Large-scale genomic and proteomic studies have divided triple-negative breast cancer into different subtypes. However, except for PARP inhibitors and cisplatin, which can be used to treat patients with mutated BRCA1 / 2 genes, no clear targeted treatment has been found for other subtypes. Various omics technologies have provided a deeper understanding of the causes and progression of triple-negative breast cancer, but the changes that occur during tumor growth and survival remain a challenge.

[0005] In addition, the diagnosis of triple-negative breast cancer lacks accurate and efficient detection markers, and clinical indicators are difficult to accurately predict the patient's prognosis, making it difficult to accurately judge the patient's prognosis and develop a reasonable personalized treatment plan. Therefore, it is necessary to find new tumor markers to improve early diagnosis, help clinicians judge the patient's prognosis and develop corresponding immunotherapy strategies. Summary of the invention

[0006] To solve the above technical problems, the present invention provides the application of MEX3D as a detection marker and therapeutic target for triple-negative breast cancer. The present invention uses the mRNA interaction capture method to establish the RNA binding protein spectrum of breast cancer, and screens out the RNA binding protein MEX3D that affects the proliferation and invasion of triple-negative breast cancer cells by bioinformatics analysis of the RNA binding protein spectrum of triple-negative breast cancer cell line MDA-MB-231 and estrogen receptor-positive breast cancer cell line MCF-7.

[0007] The first objective of the present invention is to provide the use of RNA binding protein MEX3D as a marker for detecting triple-negative breast cancer.

[0008] MEX3D is an RNA-binding protein that determines the fate of RNA from synthesis to decay, and is mainly involved in RNA shearing, modification, transport, localization, stability, translation, and degradation. The expression of RNA-binding proteins is strictly regulated, forming riboprotein complexes with target RNA through different types of domains, and regulating post-transcriptional gene expression in a variety of ways.

[0009] The second object of the present invention is to provide a triple-negative breast cancer detection kit, which uses the RNA binding protein MEX3D as a target to detect the expression or secretion of MEX3D in a sample.

[0010] Furthermore, the kit comprises a reagent for detecting the expression or secretion amount of MEX3D.

[0011] Furthermore, the kit comprises primers for amplifying MEX3D or antibodies that specifically bind to MEX3D protein.

[0012] The third object of the present invention is to provide the use of RNA binding protein MEX3D as a therapeutic target for triple-negative breast cancer.

[0013] The fourth object of the present invention is to provide a drug for treating triple-negative breast cancer, which reduces the expression of MEX3D or inhibits the activity of MEX3D protein.

[0014] The fifth object of the present invention is to provide a use of a preparation for knocking down MEX3D expression or inhibiting MEX3D protein activity in the preparation of a drug for treating triple-negative breast cancer.

[0015] Furthermore, the preparation includes shRNA.

[0016] Furthermore, the nucleotide sequence of the shRNA is shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

[0017] Further, the forward sequence of shMEX3D#1 is shown as 5'-CCGGGAGTGGTCAGGTTA CAATAAACTCGAGTTTATTGTAACCTGACCACTCTTTTTG-3' (SEQ ID NO. 1), and the reverse sequence is shown as 5'-AATTCAAAAAGAGTGGTCAGGTTACAATAAA CTCGAGTTTATTGTAACCTGACCACTC-3' (SEQ ID NO. 2).

[0018] Further, the forward sequence of shMEX3D#2 is shown as 5'-CCGGCAGCCCTTCAACGA CAGTATTCTCGAGAATACTGTCGTTGAAGGGCTGTTTTTG-3' (SEQ ID NO. 3), and the reverse sequence is shown as 5'-AATTCAAAAACAGCCCTTCAACGACAGTATT CTCGAGAATACTGTCGTTGAAGGGCTG-3' (SEQ ID NO. 4).

[0019] Beneficial effects of the present invention:

[0020] The present invention provides an application of RNA binding protein MEX3D as a detection marker and therapeutic target for triple-negative breast cancer. MEX3D is specific to triple-negative breast cancer cell lines and is highly correlated with their proliferation and invasion. It does not affect the growth of normal human cells or other types of breast cancer cells. Knocking down the expression of the RNA binding protein in the cell line can significantly block the proliferation of triple-negative breast cancer cells. MEX3D can be used as a detection marker and therapeutic target for triple-negative breast cancer, which is expected to solve the problem of the current lack of effective diagnosis and treatment methods for triple-negative breast cancer, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0022] Figure 1 This is the experimental process of capturing mRNA interacting proteins of breast cancer MDA-MB-231 and MCF-7 cells in Example 1 of the present invention;

[0023] Figure 2 The breast cancer MDA-MB-231 and MCF-7 cells in Example 1 of the present invention captured protein western;

[0024] Figure 3 It is the data analysis of the mRNA binding protein domain in Example 1 of the present invention;

[0025] Figure 4 It is the analysis of the GO results of capturing mRNA binding proteins in Example 1 of the present invention;

[0026] Figure 5 This is a graph showing the western blot results of MEX3D in different cells in Example 2 of the present invention;

[0027] Figure 6 This is a graph showing the western blot results of MEX3D in different mouse tissues in Example 2 of the present invention;

[0028] Figure 7 This is a graph showing the western blot results of MEX3D in the testes of mice of different ages in Example 2 of the present invention;

[0029] Figure 8 This is a western blot result diagram of YBX2 in different mouse tissues in Example 2 of the present invention;

[0030] Fig. 9 This is a graph showing the western blot results of YBX2 in the testes of mice of different ages in Example 2 of the present invention;

[0031] Fig.10 This is a graph showing the western blot results of YBX2 in different cells in Example 2 of the present invention;

[0032] Fig.11 is the mRNA expression of MEX3D in common tissues, non-triple-negative breast cancer cells, and triple-negative breast cancer cells in Example 3 of the present invention;

[0033] Fig.12 The growth curve of MCF-7 cells in the YBX2 knockdown group and the control group detected by the CCK-8 method in Example 4 of the present invention;

[0034] Fig.13 The growth curve after MEX3D protein knockdown in MCF-7 cells, 293T cells, and MDA-MB-231 cells was detected by CCK-8 method in Example 4 of the present invention;

[0035] Fig.14 is the cell proliferation curve after knocking down MEX3D in triple-negative breast cancer cells MDA-MB-231 in Example 4 of the present invention;

[0036] Fig.15The invasion experiment effect diagram and absorbance analysis of the triple negative breast cancer cell MDA-MB-231 cell MEX3D knockdown group and control group in Example 5 of the present invention (*0.01 <P<0.05,**0.05<P<0.01,***0.01<P<0.001);

[0037] Fig.16 is the proportion of apoptosis in each group of cells in Example 6 of the present invention (*0.01 <P<0.05,**0.05<P<0.01,***0.01<P<0.001)。 DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0039] Example 1: Establishment of a Breast Cancer RNA Binding Protein Repertoire

[0040] The mRNA interaction capture method was used to form a stable covalent bond between RNA binding proteins and RNA in triple-negative breast cancer cells MDA-MB-231 and estrogen receptor-positive breast cancer cells MCF-7. After the breast cancer cells were lysed, the RNA and RNA binding protein complex containing poly(A) tails were bound using oligo(dt)-containing magnetic beads. After elution, the obtained RNA binding proteins were subjected to gel electrophoresis and silver staining. After obtaining the target band, the captured proteins were enzymatically digested into peptides, and then analyzed and identified by liquid chromatography-mass spectrometry to obtain RNA binding protein data. The steps are as follows: Figure 1 shown.

[0041] Specific steps of mRNA interaction capture experiment:

[0042] 1. Preparation before the experiment:

[0043] 1. Sample preparation: 1×10 8 MDA-MB-231 cells were prepared and the cell pellets were stored in a -80°C refrigerator for later use. For the treatment group (cross-linking), 1×10 8 The culture medium was removed from MDA-MB-231 cells, and the cells were rinsed with PBS three times to completely remove the culture medium. The cells were then cross-linked at 254 nm using a UV cross-linker with 0.15 J / cm 2 After cross-linking, the cross-linked cells were collected with PBS, the supernatant was removed, and the cell pellet was frozen in a -80°C refrigerator for use.

[0044] 2. Buffer preparation: All buffers need to be autoclaved before adding LiDS and DTT, add LiDS and DTT after cooling, and finally filter with a 0.22um filter.

[0045] 2. RNA binding protein capture steps:

[0046] 1. Wash beads: Take 2.5 mL beads from the cross-linked group and the non-cross-linked group respectively, and gently transfer the beads to a 15 mL centrifuge tube with Lysis buffer. After the beads are combined on the magnetic rack, remove the supernatant, add 10 mL Lysis buffer to resuspend, and wash on a 360-degree shaker for 10 minutes for later use.

[0047] 2. Lysis and homogenization of cells: The cells in the cross-linking group and the non-cross-linking group were first resuspended with 5 mL of Lysis buffer, and then the cells were homogenized three times with a 3 mL syringe and a built-in needle, and then a 25G (0.5 mm) needle was used to homogenize the sample until there was no resistance. Finally, 10 mL of lysis buffer was added and mixed, and then 40 μL of lysis buffer was taken as input.

[0048] 3. Incubation of beads and lysis buffer: After combining the reserved beads on the magnetic rack, remove the supernatant, transfer to 15mL of cell lysis buffer, adjust the volume to 35mL, and incubate on a 360-degree shaker in a 4°C refrigerator for 1h. Place the 50mL centrifuge tube containing the lysis buffer and beads on the magnetic rack, and you can observe that the beads are slowly adsorbed on one side of the magnetic rack, which takes about 30min.

[0049] 4. Aspirate beads-RNA-protein complex:

[0050] (1) Remove the supernatant, add 5 mL of pre-cooled Lysis buffer, gently pipette to resuspend with a 1 mL pipette tip, adjust the volume to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and bind on a magnetic stand for 10-15 min until clarified;

[0051] (2) Remove the supernatant, add 5 mL of pre-cooled Wash buffer 1, gently pipette to resuspend with a 1 mL pipette tip, adjust the volume to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and combine on a magnetic stand for 10-15 min until clarified;

[0052] (3) Remove the supernatant, add 5 mL of pre-cooled Wash buffer 1, gently pipette to resuspend with a 1 mL pipette tip, adjust the volume to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and combine on a magnetic stand for 10-15 min until clarified;

[0053] (4) Remove the supernatant, add 5 mL of pre-cooled Wash buffer 2, gently pipette to resuspend with a 1 mL pipette tip, adjust the volume to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and combine on a magnetic stand for 10-15 min until clarified;

[0054] (5) Remove the supernatant, add 5 mL of pre-cooled Wash buffer 2, gently pipette to resuspend with a 1 mL pipette tip, adjust the volume to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and combine on a magnetic stand for 10-15 min until clarified;

[0055] (6) Remove the supernatant, add 5 mL of pre-cooled Low Salt buffer, gently pipette to resuspend with a 1 mL pipette tip, dilute to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and bind on a magnetic stand for 10-15 min until clarified;

[0056] (7) Remove the supernatant, add 5 mL of pre-cooled Low Salt buffer, gently pipette to resuspend with a 1 mL pipette tip, adjust the volume to 35 mL, incubate on a 360-degree shaker in a 4°C refrigerator for 5 min, and bind on a magnetic rack for 10-15 min until clarified.

[0057] 5. Elution of RNA and protein:

[0058] (1) Transfer the beads complex to a 1.5 mL EP tube with 1 mL low salt buffer, adsorb with a magnetic stand, and remove the supernatant;

[0059] (2) Add 500 μL of elution buffer and incubate at 55°C for 3 min. Collect the supernatant after adsorption on a magnetic rack. Repeat this step to obtain 1 mL of RNA and protein complex.

[0060] The binding protein capture results are as follows Figure 2 shown.

[0061] 6. Take the corresponding amount of sample for silver staining and mass spectrometry identification:

[0062] Silver staining reagents include acetic acid, anhydrous ethanol, sodium thiosulfate, anhydrous sodium acetate, silver nitrate, anhydrous sodium carbonate, glycine and formaldehyde.

[0063] Silver staining main experimental steps:

[0064] (1) After gel electrophoresis, the samples were rinsed twice with double distilled water;

[0065] (2) Fixation for 1 h: 10 mL of acetic acid, 40 mL of anhydrous ethanol, and double distilled water to 100 mL;

[0066] (3) Wash once: rinse the gel with double distilled water for 1 min;

[0067] (4) Sensitization for 30 min: 30 mL of anhydrous ethanol, 0.2 g of sodium thiosulfate, 6.8 g of anhydrous sodium acetate, and double distilled water to make up to 100 mL;

[0068] (5) Wash with water three times, 5 minutes each time;

[0069] (6) Silver staining for 20 min: 0.25 g silver nitrate, dilute to 100 mL with double distilled water (avoid light and add 20 μL formaldehyde before use);

[0070] (7) Wash once with water for 2 minutes (avoid light);

[0071] (8) Color development for 3-5 min: 2.5 g of anhydrous sodium carbonate, dilute to 100 mL with double distilled water (add 10 μL of formaldehyde before use);

[0072] (9) 15 min after termination: 0.4 g glycine, dilute to 100 mL with double distilled water;

[0073] (10) Storage: 10% ethanol, 4°C. Cut the gel and send part of it for mass spectrometry.

[0074] After filtering out nonspecific RNA binding proteins in the data, PFAM analysis and comparison were performed on the RNA binding proteins, and the main structural features of RNA binding proteins in MDA-MB-231 and MCF-7 cells were obtained (such as Figure 3 ) containing RRM domain, DEAD domain, and non-classical Helicase C and classical KH, ZF-CCCH and other RNA binding domains; GO analysis was also performed on these data, and it was found that they have functions related to affecting gene transcription. The results are as follows Figure 4 As shown, for example: rRNA, mRNA binding ability, poly (A) tail RNA binding ability, nucleotide binding ability, ribosome protein components, ATP-independent RNA helicase activity, general helicase activity and other properties.

[0075] The breast cancer-related proteins in the TCGA database data were analyzed for structure and correlation with RBP (RNA-Binding Protein) in mass spectrometry analysis, and two proteins that met the screening criteria were obtained, namely MEX family protein MEX3D and Y box family protein YBX2.

[0076] Example 2: Expression differences of MEX3D and YBX2 in cells, tissues and mouse testis at different ages

[0077] To verify whether MEX3D and YBX2 can be used as targets for the detection and treatment of triple-negative breast cancer, proteins were extracted from 293T, MDA-MB-231, MCF-7, MCF-10A, Hela cells, as well as heart, liver, lung, kidney, testis, brain tissue, muscle, intestine and other tissues for comparison. It was found that MEX3D was expressed in MDA-MVB-231 cells (e.g. Figure 5 As shown in Figure 6 ), and by verifying the protein expression in testicular tissue at multiple weeks of age, we found that MEX3D began to be highly expressed in the testis of 35-day-old mice, and its expression was maintained until adulthood (as shown in Figure 7 shown).

[0078] YBX2 protein is highly expressed in mouse testis (e.g. Figure 8 ), and this high expression persisted in the testes of mice aged 7-24 days (as shown in Fig. 9 However, YBX2 was only highly expressed in MCF-7 cells, and did not show a difference in expression in triple-negative breast cancer cells MDA-MVB-231 (as shown in Fig.10 This indicates that YBX2 cannot be used as a detection or therapeutic target for triple-negative breast cancer cells.

[0079] The above experiments verified that MEX3D is an RBP highly expressed in testis / tumor (C / T). This type of gene is highly expressed in testis and tumors of different tissue origins. This tissue expression pattern supports that these genes may be targets for active specific immunotherapy.

[0080] Example 3: Differences in mRNA expression of MEX3D

[0081] The mRNA expression of MEX3D in normal tissues, non-triple-negative breast cancer cells, and triple-negative breast cancer cells was detected. Fig.11 As shown, MEX3D mRNA expression was significantly expressed in triple-negative breast cancer cells, further verifying that MEX3D can be used as a detection marker for triple-negative breast cancer.

[0082] Example 4: Effect of mRNA binding protein on breast cancer cell proliferation

[0083] In order to further verify the effect of mRNA binding proteins on the proliferation ability of breast cancer cells, we constructed shRNAs of MEX3D and YXB2 and treated MCF-7, MDA-MB-231, and 293T cells respectively, and then used CCK-8 method to measure the growth curves. Fig.12 and Fig.13As shown in Figure 2, in MDA-MB-231 cells treated with shMEX3D, the cell proliferation ability was significantly weakened (e.g. Fig.14 The proliferation ability of cells was not changed after knocking down MEX3D in 293T cells and MCF-7 cells. The proliferation ability of cells was not changed after shYBX2 treatment.

[0084] The forward sequence of shMEX3D#1 is shown as 5′-CCGGGAGTGGTCAGGTTACAATAAAC TCGAGTTTATTGTAACCTGACCACTCTTTTTG-3′ (SEQ ID NO. 1), and the reverse sequence is shown as 5′-AATTCAAAAAGAGTGGTCAGGTTACAATAAACTCGAGT TTATTGTAACCTGACCACTC-3′ (SEQ ID NO. 2).

[0085] The forward sequence of shMEX3D#2 is shown as 5′-CCGGCAGCCCTTCAACGACAGTATTC TCGAGAATACTGTCGTTGAAGGGCTGTTTTTG-3′ (SEQ ID NO. 3), and the reverse sequence is shown as 5′-AATTCAAAAACAGCCCTTCAACGACAGTATTCTCGAGA ATACTGTCGTTGAAGGGCTG-3′ (SEQ ID NO. 4).

[0086] The forward sequence of shYBX2#1 is shown as 5'-CCGGGCGCAGAAGCCACTAATGTAACTCGAGTTACATTAGTGGCTTCTGCGCTTTTTG-3' (SEQ ID NO. 5), and the reverse sequence is shown as 5'-AATTCAAAAAGCGCAGAAGCCACTAATGTAACTCGAGTTA CATTAGTGGCTTCTGCGC-3' (SEQ ID NO. 6).

[0087] The forward sequence of shYBX2#2 is shown as 5'-CCGGGCGCAGAAGCCACTAATGTAACTCGAGTTACATTAGTGGCTTCTGCGCTTTTTG-3' (SEQ ID NO. 7), and the reverse sequence is shown as 5'-AATTCAAAAAGCGCAGAAGCCACTAATGTAACTCGAGTTA CATTAGTGGCTTCTGCGC-3' (SEQ ID NO. 8).

[0088] 1. Construction of PLKO.1-shMEX3D / PLKO.1-shYBX2 vector

[0089] (1) Primer design website: http: / / www.broadinstitute.org / rnai / public / seq / search;

[0090] (2) Order shMEX3D and shYBX2 oligo primers;

[0091] (3) The oligonucleotide chains were annealed into double strands. The reaction system was as follows: 4.5 μL Forward oligo (100 μM), 4.5 μL Reverse oligo (100 μM), 1 μL NEB buffer 2; the PCR was performed in a PCR instrument according to the following program: 95°C, 5 min, 95-85°C at -2°C / s, 85-25°C at -0.1°C / s, hold at 4°C;

[0092] (4) Enzyme digestion of PLKO.1-TRC cloning vector, the reaction system is as follows: 1 μg PLKO.1-TRC cloning vector, 5 μL NEBCut Smart buffer, 1 μL EcoR I, 1 μL Age I, sterile water to 50 μL, incubate in a 37°C water bath for 2-3 h;

[0093] (5) Electrophoresis: Prepare 1% agarose gel, load all the enzyme-digested samples, and run electrophoresis at 120V for 1 h. Two bands are visible after exposure, one about 7 kb and the other about 1.9 kb. Cut the gel and recover the 7 kb band into a 1.5 mL EP tube;

[0094] (6) Purify DNA using a gel recovery kit and measure the DNA concentration;

[0095] (7) Ligation, the reaction system is as follows: 2 μL of annealed oligonucleotide double strands, 50 ng of PLKO.1 vector recovered by restriction digestion, 2 μL of T4 DNA ligase buffer, 1 μL of T4 ligase, add sterile water to make up to 20 μL system, 16°C, 4-20 h;

[0096] (8) Transformation: Remove DH5α competent cells from -80°C and place on ice to thaw; add 10 μL of the ligation product to 100 μL of DH5α competent cells, gently flick to mix, and place on ice for 30 min; heat shock in a 42°C metal bath for 45-90 s, remove quickly and place on ice for 2 min;

[0097] (9) Add 900 μL of LB medium without antibiotics and incubate at 37°C, 225 rpm on a shaker for 45-60 min;

[0098] (10) Take 100 μL of the competent cell suspension after transformation and evenly spread it on the LB solid plate medium containing ampicillin antibiotics using a sterile spreading stick (sterilized by burning with an alcohol lamp in advance);

[0099] (11) Place the LB solid culture medium upside down in a 37°C biochemical incubator and culture for 12-16 hours;

[0100] (12) Pick 4-6 single clones and inoculate them together with the pipette tip into fresh LB liquid medium containing ampicillin and culture for 12-16 h;

[0101] (13) Extract the plasmid and send it to the company for sequencing;

[0102] (14) According to the sequencing results, the correctly sequenced PLKO.1-shMEX3D / PLKO.1-shYBX2 was selected.

[0103] 2. Lentivirus Packaging

[0104] (1) Prepare the following experimental reagents: 2 M CaCl2, 2×HeBS pH 7.05, permeabilizing agent polybrene (10 mg / mL), screening agent puromycin (10 mg / mL), DMEM medium, FBS, PBS, and 0.25% trypsin;

[0105] (2) HEK-293T cell culture steps: Take out HEK-293T cells grown to 80% from the incubator; remove the culture medium and wash once with 5 mL PBS; add 1 mL of trypsin for 30 seconds, and add 5 mL of complete culture medium (containing 10% FBS, 1% double antibody) to stop digestion; transfer the cells to a 15 mL centrifuge tube, centrifuge at 300g for 5 minutes to remove the trypsin; add 1 mL of fresh complete culture medium to resuspend the cells; take a 6-well plate and add 2 mL of culture medium to each well; take 100 μL of the resuspended cells and drop them into the 6-well plate, mix well, and return to the incubator to culture for about 12 hours;

[0106] (3) CaCl2 method to transfect cells to obtain large amounts of virus

[0107] Prepare plasmid solution in a 1.5 mL EP tube: X μL sterile water, 2.5 μg plasmid (1 μg pSPAX2 + 0.5 μg pM2.G + 1 μg PLKO.1-shRNA), 50 μL 0.5 M CaCl2 (diluted with 2 M CaCl2), X + plasmid volume + 50 μL = 100 μL;

[0108] Mix the required volume of HeBS on a vortexer at 300 rpm, add 100 μL of HeBS buffer to the plasmid suspension, mix gently 2-3 times with a 1 mL pipette, and leave at room temperature for 20 min.

[0109] The HEK-293T cell culture medium was replaced with a culture medium without dual antibodies (DMEM + 10% FBS);

[0110] Slowly add the mixture to 293T cells, gently shake the six-well plate after adding, and gently mix (black particles can be seen in the cells 20 minutes after plasmid transfection);

[0111] Culture overnight in a 37°C incubator. Change the medium (DMEM + 10% FBS) as early as possible the next morning and continue culturing for 48 hours to obtain a large amount of virus;

[0112] (4) Virus collection: Filter the virus through a 0.45 μm filter, dispense 500 μL of the virus into tubes, and store in a -80°C refrigerator;

[0113] 3. Lentiviral transfection of cells:

[0114] (1) 293T, MDA-MB-231 and MCF-7 cells were plated in 6-well plates. When the cell density reached 60-70%, 350 μL of virus was added to each well, and 1.88 μL of permeabilization agent polybrene (working concentration 8 μg / mL) was added at the same time. The cells were cultured in a 37°C incubator overnight. The medium was changed as early as possible on the next morning. On the morning of the third day, the selection agent (mother solution 10 mg / mL, working concentration 1 μg / mL) was added to the infected 293T cells, MDA-MB-231 cells and MCF-7 cells. The cells were usually incubated for 2-3 days.

[0115] (2) Take some cells to extract RNA for real-time quantitative PCR to verify the knockdown efficiency.

[0116] Example 5: Effect of MEX3D on the invasion ability of triple-negative breast cancer cells

[0117] Tumor invasion is a complex process involving multiple molecules, including changes in cell adhesion, secretion of matrix-degrading enzymes, enhanced motility, invasion of blood vessels into the circulation, reaching secondary sites to form clones, and clone proliferation to form metastatic foci. The characteristic of tumors that are prone to metastasis is the fundamental reason why radiotherapy and chemotherapy are difficult to fundamentally cure malignant tumors, often leading to consequences such as tumor recurrence, poor prognosis, and patient death. The Transwell invasion experiment mimics the process of tumor invasion in vivo. We spread a layer of matrix gel on the surface of the upper chamber of the transwell filter, inoculated tumor cells in the upper chamber of the transwell containing matrix gel, and detected the number of cells that passed through the matrix gel to the back of the transwell filter after 36-48 hours. Through the Transwell invasion experiment, the effect of MEX3D on the invasion ability of MDA-MB-231 cells was further verified.

[0118] Compared with the normal control group, the cell invasion ability of MDA-MB-231 cells with MEX3D knockdown was significantly reduced. The cells on the transwell filter membrane were lysed with RIPA lysis buffer, and the absorbance analysis of the lysate at a wavelength of 570nm showed that the OD value of the MEX3D knockdown group was significantly lower than that of the control group. The knockdown of MEX3D had a significant weakening effect on the invasion and metastasis of MDA-MB-231 cells ( Fig.15 ).

[0119] Example 6: Effect of MEX3D on apoptosis of triple-negative breast cancer cells

[0120] Cell apoptosis was analyzed by flow cytometry. Fig.16 As shown, it was found that compared with the normal control group, the number of apoptotic MDA-MB-231 cells in the cells with MEX3D knockdown was significantly increased, and the knockdown of MEX3D promoted the apoptosis of MDA-MB-231 cells.

[0121] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A drug for treating triple-negative breast cancer, characterized in that: The drug targets the RNA binding protein MEX3D, knocks down the expression of MEX3D or inhibits the activity of the MEX3D protein. The drug includes shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

2. Use of a preparation for knocking down MEX3D expression or inhibiting MEX3D protein activity in the preparation of a drug for treating triple-negative breast cancer, the preparation comprising shRNA, the nucleotide sequence of the shRNA being shown in SEQ ID NO.1-2 or SEQ ID NO.3-4.

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