CN116377061B - New auxiliary chemotherapy drug resistance marker for breast cancer and application thereof - Google Patents

Abstract

The invention discloses a breast cancer neoadjuvant chemotherapy resistance marker and its application. The present invention determines the chemotherapy resistance and prognosis of breast cancer patients by detecting the expression of CD96 in tumor cells, and can promote the sensitivity of breast cancer cells to chemotherapy drugs by targeting CD96. Through molecular biology research on clinical breast cancer tissue specimens, cohort follow-up data and in vitro cell models in their own work, the present invention found that CD96, which is expressed on the surface of immune cells, is highly expressed in breast cancer cells or breast cancer cell lines of some patients. And it is positively correlated with the poor prognosis of breast cancer after chemotherapy; further immunofluorescence and flow cytometry experiments showed that CD96 inhibits the sensitivity of breast cancer cells to neoadjuvant chemotherapy drugs, and knocking down the CD96 ligand CD155 can inhibit the CD96 With the same chemotherapeutic effect, the binding site of mutated CD96 and CD155 can significantly inhibit the drug resistance regulatory effect of CD96.

Description

Breast cancer neoadjuvant chemotherapy resistance markers and their applications

Technical field

The invention relates to a neoadjuvant chemotherapy resistance marker and its application, which belongs to the field of biomedical technology, and particularly relates to the breast cancer neoadjuvant chemotherapy resistance marker CD96 and its use in predicting the neoadjuvant chemotherapy resistance and prognosis of breast cancer. application.

Background technique

Breast cancer is the number one malignant tumor in women. Although the current treatment options can maintain the 10-year overall survival rate of early-stage breast cancer patients at more than 80%, most patients have large tumors or metastasis at diagnosis, which brings great difficulties to surgery. These major problems lead to unsatisfactory treatment results and ultimately bring a heavy economic burden to families and society.

Neoadjuvant chemotherapy is one of the main ways to treat breast cancer. Its advantages are: first, it can eliminate tiny metastatic lesions and reduce the chance of intraoperative dissemination. Secondly, it can shrink the tumor, downstage it, and improve the resection rate of radical surgery. Thirdly, the sensitivity of the tumor to chemotherapy drugs can be judged.

However, there are many options for neoadjuvant therapy, and there is currently no corresponding solution for predicting whether patients are sensitive to these drugs, which delays patients' treatment opportunities and provides more opportunities for tumor progression.

Therefore, it is particularly important to predict treatment-resistant patients as early as possible and provide alternative interventions.

Contents of the invention

The purpose of the present invention is to provide a new marker CD96 that can predict breast cancer chemotherapy resistance, judge the chemotherapy resistance and prognosis of breast cancer patients by detecting the expression of CD96 in tumor cells, and can promote breast cancer by targeting CD96 Sensitivity of cancer cells to chemotherapy drugs.

The purpose of the present invention and solving its technical problems are achieved by adopting the following technical solutions.

The present invention provides the application of a reagent for detecting CD96 expression levels in preparing products for predicting neoadjuvant chemotherapy resistance and prognosis of breast cancer.

Furthermore, the reagent for detecting the expression level of CD96 includes primers for detecting the expression level of the CD96 gene.

Furthermore, the primers for detecting the expression level of CD96 gene are as shown in SEQ ID NO. 1-2.

The invention provides a kit for predicting the resistance and prognosis of neoadjuvant chemotherapy in breast cancer. The kit includes a reagent for detecting the expression level of CD96.

Furthermore, the reagents for detecting the expression level of CD96 include reagents for detecting primers for detecting the expression level of the CD96 gene; the primers for detecting the expression level of the CD96 gene are as shown in SEQ ID NO. 1-2.

Furthermore, the kit includes one or more of nucleic acid extraction reagents, PCR reagents, gene-specific primers or probes.

The present invention provides the application of CD96 inhibitors in the preparation of products that improve the sensitivity of neoadjuvant chemotherapy for breast cancer.

Furthermore, the CD96 inhibitor is selected from CD96 neutralizing antibodies, and/or shRNA designed based on CD96, and/or sgRNA designed based on its upstream regulatory gene CD155, and/or mutated CD155 and the 75th amino acid of CD96 binding site reagent.

Furthermore, the shRNA designed based on CD96 is shown in SEQ ID NO.3 and/or SEQ ID NO.4; the target sequence of the gene CD155 is shown in SEQ ID NO.5; the shRNA based on its upstream The sgRNA designed to regulate the gene CD155 is shown in SEQ ID NO. 6-7; the sequence of the binding site of the 75th amino acid between the mutant CD155 and CD96 is shown in SEQ ID NO. 8.

The present invention provides the application of CRISPR CAS9 based on CD155 designed sgRNA in the preparation of products that improve the sensitivity of neoadjuvant chemotherapy in breast cancer.

Through the above technical solutions, the present invention has at least the following advantages:

1) A neoadjuvant chemotherapy resistance marker of the present invention, specifically CD96. Through molecular biology research on clinical breast cancer tissue specimens, cohort follow-up data and in vitro cell models in my own work, I found that CD96 is highly expressed in breast cancer cells or cell lines in some patients and is positively correlated with poor prognosis after breast cancer chemotherapy. ; Further immunofluorescence and flow cytometry experiments showed that CD96 inhibits the sensitivity of breast cancer cells to neoadjuvant chemotherapy drugs. Knocking down the CD96 ligand CD155 can have the same chemopromoting effect as CD96 inhibition. The difference between mutant CD96 and CD155 The binding site can significantly inhibit the drug resistance regulatory effect of CD96.

2) CD96 activates tumor cell resistance to neoadjuvant chemotherapy drug treatment by binding to CD155. Research on this marker has found that on the one hand, in-depth research can identify the relationship between the CD96 pathway in regulating tumor resistance and provide scientific basis for new ways to target this pathway to treat breast cancer; on the other hand, it can also identify people who are resistant to neoadjuvant chemotherapy. Provide prompts and play a role in personalized treatment and precise treatment in breast cancer treatment or other cancer treatments.

Description of the drawings

Figure 1 shows the expression results of CD96 in tissue sections of breast cancer patients in Example 1;

Figure 1a shows the expression of CD96 in tumor cells in tumor specimens after breast cancer surgery using tissue immunofluorescence technology;

Figure 1b shows the expression of CD96 in tumor cells in tumor specimens after breast cancer surgery using immunohistochemistry;

Figure 1c shows the proportion of breast cancer patients with high CD96 expression and low CD96 expression.

Figure 2 shows the expression results of CD96 in different breast cancer cell lines in Example 1;

Figure 2a shows the expression of CD96 in different breast cancer cell lines detected by q-PCR technology;

Figure 2b shows the expression of CD96 in different breast cancer cell lines detected by Western blotting technology;

Figure 2c shows the expression of CD96 in MCF-7 and MDA-MB-468 breast cancer cells detected by cellular immunofluorescence technology;

Figure 2d shows the expression of CD96 in different breast cancer cell lines detected by flow cytometry.

Figure 3 shows the effect of CD96 expression in breast cancer cells and patient survival rate in Example 1.

Figure 4 shows the effect of CD96 on the survival rates of patients with different subtypes of breast cancer in Example 1.

Figure 5 is an analysis of the relationship between the expression of CD96 in cancer cells and the effect of neoadjuvant chemotherapy in patients in Example 2.

Figure 6 shows the relationship between CD96 and TUNEL in tumor cells in patients after neoadjuvant chemotherapy in Example 2;

Figure 6a shows the co-localization of CD96 and the apoptosis marker TUNEL in tumor cells using immunofluorescence technology;

Figure 6b shows the correlation between CD96 and TUNEL in tumor cells of 20 patients after breast cancer surgery.

Figure 7 shows the effect of CD96 neutralizing antibody in in vitro chemotherapy in Example 2;

Figure 8 shows the efficiency detection of CD96 gene knockout in Example 2;

Figure 9 shows the effect of CD96 gene knockout on in vitro chemotherapy in Example 2;

Figure 10 is the detection of the efficiency of CD155 knockout by CRISPR-CAS9 in Example 3;

Figure 11 shows the effect of combined use of CD155 knockout and CD96 neutralizing antibody on in vitro chemotherapy in Example 3;

Figure 12 shows the effect of chemotherapy on tumor cells after mutating the binding site of CD96 and CD155 in Example 3.

Detailed ways

Example 1 Study on the expression level of CD96 in breast cancer and its relationship with the prognosis of breast cancer patients

1. Detection of CD96 expression in breast cancer

1.1 Immunofluorescence technology: Randomly select tissue sections from 616 patients who have undergone chemotherapy and surgery. First, bake the sections in an oven at 60°C for 20 minutes and dewax them; then soak them in xylene twice, 10 minutes each time. Hydration: soak in 100% alcohol twice, 10 minutes each time; gradient alcohol soak: 95%, 90%, 80%, 70%, 60%, 50%, 5 minutes each time. Antigen retrieval: Prepare pH8.0 EDTA, then soak in it, put it in a pressure cooker and boil for 2 minutes for repair. After the liquid drops to normal temperature, 5% BSA is used to block the antigen for 20 minutes, and then gently shaken dry. Add CD96 primary antibody (Thermo Company, product number PA5-97568, 479; concentration is 1:150 dilution) and tumor marker Cytokeratin primary antibody ( Abcam Company, Cat. No. MNF116, concentration: 1:100 dilution), incubate at 4°C overnight. Equilibrate at room temperature for 30 minutes, discard the primary antibody; soak in PBS 2 to 3 times, 5 minutes each time. The tissue sections were successively incubated with immunofluorescent secondary antibodies of the corresponding species and DAPI (for 1 hour at room temperature); soaked in PBS 2 to 3 times, 5 min each time. The slides were mounted with anti-fluorescence quenching agent and recorded with Zeiss 800. Finally, the expression of CD96 on the surface of tumor cells in the sections was recorded; the relevant data were analyzed.

1.2 Immunohistochemistry: Section dewaxing, antigen retrieval and primary antibody staining are the same as immunofluorescence; after primary antibody cleaning, add secondary antibodies, incubate at room temperature for 30 minutes, and soak in PBS 2 to 3 times, 5 minutes each time. DAB staining (according to the color development effect under the microscope), shake off the dyeing solution, soak in PBS to terminate the staining; soak the hematoxylin stained nuclei, wash away the hematoxylin with tap water, and recover the hematoxylin; rinse slowly with slow water, spin dry at 37°C, and dry at 37°C with medium Seal the slides with plastic resin and blow dry in a fume hood to complete the production.

1.3 Construct the PCR primer detection kit and RT-PCR detection of CD96: synthesize the upstream and downstream primers of CD96, the sequences are: upstream (SEQ ID NO.1): CATCCCCAATACGGCTTCTA; downstream (SEQ ID NO.2): TGCTGTTCCATTCATCTGC. The cells were digested into single cells by 0.25% trypsin, then washed twice with PBS, and digested with 1 ml TRIzol for 20 minutes. Add 0.2ml chloroform for every 1ml of TRIzol used, shake vigorously for 15 seconds, and leave at room temperature for 3 minutes. Centrifuge at 10,000 × g for 15 minutes at 2-8°C.

The sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase and an intermediate layer. RNA is mainly in the aqueous phase, and the volume of the aqueous phase is approximately 60% of the TRIzol reagent used. Transfer the aqueous phase to an RNase-free EP tube, precipitate the RNA in the aqueous phase with isopropyl alcohol (add 0.5 ml of isopropyl alcohol for every 1 ml of TRIzol used), and leave it at room temperature for 10 minutes. Centrifuge again at 10,000 × g for 10 min at 2-8°C. Remove supernatant. Wash the RNA pellet with 75% ethanol (add at least 1 ml of 75% ethanol for every 1 ml of TRIzol used). Centrifuge at no more than 7500 × g for 5 minutes at 2-8°C and discard the supernatant. Let the RNA pellet dry at room temperature. Add 25-200 μl of RNase-free water to dissolve the RNA. Use Oligo(dT) 18 and the reaction conditions are 50°C for 45 minutes, and then 85°C for 5 minutes to inactivate the reverse transcriptase and reverse-transcribe RNA into cDNA.

PCR reaction detects the expression of CD96. The reaction system is as follows:

17.5ul DEPC water, 2.5ul 10×Taq buffer, 2.0ul MgCl ₂ , 0.5ul 10M dNTP Mix, 0.5ul upstream primer, 0.5ul downstream primer, 0.5ul Tap enzyme (5u/ul), 1.0ul cDNA. Amplification is performed in a qPCR instrument through DNA denaturation (90°C-96°C), annealing (25°C-65°C) and extension.

1.4 Western blotting was used to detect the expression of CD96 in SKBR3, BT-474, MCF-7, MDA-MB-361, BT-549, MDA-MB-468, and MDA-MB-231 breast cancer cell lines.

1.5 Cell immunofluorescence detection of CD96 expression in MCF-7 and MDA-MB-468 breast cancer cell lines.

1.6 Detect the expression of CD96 in cell lines by flow cytometry; digest the cells with 0.25% trypsin, wash them twice with PBS, resuspend the cells and count them, resuspend the cells with cell wash (PBS containing 2% BSA) to block the antigen, Make the cell concentration 2×10 ⁶ /mL; incubate with CD96 primary antibody (Biolegend Company, Cat. No. 338410 1:1000): add IgG1, κ isotype channel antibody (Biolegend Company, Cat. No. 400121 1:1000) to the control tube, and mix thoroughly , incubate at 4°C for 30 minutes. During the incubation period, shake the reaction tube every 10 minutes to allow the cells and antibodies to fully react; then wash the cells 3 times with PBS, centrifuge at 1000 rpm for 5 minutes each time, discard the supernatant, resuspend the cells and put them on the machine for detection ( Cytoflex).

2. Relationship between CD96 expression level and prognosis of breast cancer patients

Two pathologists were invited to score the CD96 immunohistochemistry results of breast cancer sections, and the product of expression area (0-4 points) and expression intensity (0-3 points) was recorded as the total score. ImageJ was used to analyze the proportion of CD96-positive tumor cells in the sections, and the positive proportion was multiplied by the immunohistochemistry score. The obtained values were stratified by X-tile and divided into high-expression and low-expression groups. Survival analysis was performed using the Kaplan-Meier method, assisted by GraphPad Prism7.0 software.

result:

1.1 Immunohistochemistry and immunofluorescence detection show that CD96 is expressed on the surface of tumor cells in sections of some tumor patients. Analysis by X-tile software showed that 38.3% of patients had high expression of CD96 (Figure 1).

1.2 RT-PCR, Western blot, immunofluorescence and flow cytometry results show that CD96 is highly expressed in some breast cancer cell lines, especially in triple-negative breast cancer cells with high malignancy and strong drug resistance. Enhancement (Figure 2). The results of these experiments are consistent, indicating that the PCR primers constructed by the applicant are correct and proven, and are an effective kit.

1.3 The tumor cells in 236 of the 616 samples included in this example highly expressed CD96. Breast cancer patients with high expression of CD96 in tumor cells have significantly worse overall survival rates and disease-free survival rates (Figure 3). High expression of CD96 in tumor cells also has a significantly worse prognosis in most molecular subtypes and stages of breast cancer (Figure 4).

The above results show that the expression level of CD96 in cancer cells in breast cancer tissue is positively correlated with the patient's pathological grade, clinical stage and poor prognosis.

Example 2 Study on the relationship between CD96 and the regulation of neoadjuvant chemotherapy resistance

1. The relationship between neoadjuvant chemotherapy and CD96 expression in cancer cells in clinical breast cancer patients

20 untreated breast patients were collected, and the tumor tissue was confirmed by hollow needle puncture. They underwent 4-6 courses of conventional neoadjuvant chemotherapy containing docetaxel. The remaining lesions were surgically removed, embedded and fixed in paraffin, and sectioned. (5-6μm), followed by dewaxing, hydration, antigen retrieval, and immunofluorescence staining for CD96, Cytokeratin, and apoptosis marker TUNEL (see above for methods). GraphPad Prism7.0 and ImageJ were used to analyze the ratio of CD96 and Cytokeratin double-positive cells to TUNEL and Cytokeratin double-positive cells and the relationship between the two.

The changes in the patient's tumor size before and after chemotherapy are evaluated through magnetic resonance results and tested according to the patient's clinical prognosis evaluation standards. Complete Response (CR) refers to the complete disappearance of the tumor, and Partial Response (PR) refers to the reduction of the maximum diameter of the tumor by more than 30%. , Progressive Disease (PD) refers to an increase in tumor diameter of more than 20%, and stable disease (SD) refers to a state between partial remission and disease progression. CR and PR are defined as chemotherapy sensitivity, and PD and SD are defined as tumor chemotherapy resistance.

2. Research on CD96’s involvement in tumor chemotherapy resistance in tumor cells

2.1 Flow cytometry was used to detect the apoptosis changes of tumor cells after CD96 intervention. Annexin V Apoptosis Detection Kit (BioLegend Company, Cat. No. 640932) was used for apoptosis detection. The specific steps are as follows:

After MDA-MB-231 breast cancer cells were cultured to 70% density, they were treated with 0.02 μg/mL docetaxel combined with 50 μg/mL CD96 neutralizing antibody for 24 hours. The cell culture medium was sucked out into a suitable centrifuge tube and washed with PBS. Once the parietal cells are removed, add an appropriate amount of EDTA-free trypsin cell digestion solution to digest the cells (EDTA may affect the binding of Annexin V to phosphatidylserine). Incubate at room temperature until the adherent cells can be knocked down by gentle pipetting, and then aspirate the trypsin cell digestion solution. Overdigestion by pancreatic enzymes needs to be avoided. Add the collected cell culture medium, gently pipet down the cells, transfer to a centrifuge tube, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count. Take 50,000 to 100,000 resuspended cells, centrifuge at 1,000g for 5 minutes, discard the supernatant, and add 195 μl of Annexin V binding solution to gently resuspend the cells. Add 5 μl Annexin V and mix gently for 15 min. Add 10 μl propidium iodide staining solution, mix gently, incubate at room temperature in the dark for 10-20 minutes, then place in an ice bath, immediately run on the machine for detection, and complete the detection within 1 hour.

2.2 Construction of CD96 knockout cell lines:

A CD96 knockdown breast cancer cell line (MDA-MB-231) was constructed through a lentiviral transfection system, using nonsense sequence and empty vector as negative controls. 48 hours after cell transfection, CD96 expression changes were detected by Western blotting for verification. Lentivirus construction: The pLKO.1 vector (Sigma Company) was double digested with restriction endonucleases ageⅠ and EcoRⅠ. The digested products were recovered with low melting point agarose gel and mixed with the annealed products of the above oligonucleotides. , and then ligated with T4 DNA ligase overnight.

The sequence of ShCD96 is: Sh-CD96-1 (SEQ ID NO.3): CCAACGAAAGTGATCTGCC; Sh-CD96-2 (SEQ ID NO.4): AGTGGAAGGTACGAGTGTA; the connected recombinant plasmid was transformed into competent DH5α bacteria (Takara Company), and After screening positive clones in ampicillin medium, single colonies were picked for expansion and culture. Plasmids were extracted using the Omega kit and further identified by DNA sequencing. The shCD96 plasmid and packaging plasmid were co-transfected with Lipofectamine 3000 (Thermo Company) into 293T cells. Cell supernatants were collected 24 hours and 48 hours after transfection and then used to infect MDA-MB-231 cells respectively.

2.3 TUNEL fluorescence staining was used to detect cell apoptosis. The cells were seeded into a 24-well plate with sterile coverslips. When the cells grew to 60%, 0.02 μg/mL docetaxel combined with 50 μg/mL CD96 neutralizing antibody was administered ( Antibodies-online Company, Cat. No. AA321-519) will be processed within 24 hours. Cells were washed twice with trypsin and washed twice with PBS. Cells were stained with TUNEL (Roche, Cat. No. 11684817910) at a concentration of 1:300 and DAPI (1:200) at 37°C for 30 minutes. The cells were washed twice with PBS and the coverslips were sealed with anti-fluorescence quenching agent. Zeiss 800 was used to observe and photograph cells.

result:

1.1 Using patient clinical efficacy evaluation criteria to evaluate 540 breast cancer patients after chemotherapy, it was found that high expression of CD96 in tumor cells significantly increased the tolerance of patients to conventional neoadjuvant chemotherapy regimens containing docetaxel. It shows that CD96 inhibits the therapeutic effect of chemotherapy drugs on tumors (Figure 5).

1.2 In the tumor specimens of 20 new breast cancer patients after chemotherapy, when detecting the apoptosis of tumor cells, it was found that CD96 and TUNEL were inversely proportional in cells positive for the tumor cell marker Cytokeratin, further indicating that CD96 inhibits the killing effect of chemotherapy drugs on tumor cells. (Figure 6).

1.3 Annexin V flow cytometry and TUNEL staining revealed that MDA-MB-231 cells can significantly promote the killing effect of neoadjuvant chemotherapy on tumor cells after inhibiting the CD96 pathway through CD96 neutralizing antibodies (Figure 7).

1.4 After CD96 is knocked out by shRNA, the expression of CD96 in cells can be significantly inhibited, proving that the knockout efficiency is reliable (Figure 8).

1.5 CD96 knockout promotes the killing of tumor cells by MDA-MB-231 cells under neoadjuvant chemotherapy (Figure 9), indicating that CD96 is an important protein that can reliably predict prognosis and tumor drug resistance.

The above results indicate that CD96 is involved in the regulation of drug resistance of tumor cells in vivo and in vitro.

Example 3 Study on CD96 ligand, CD155, participating in the regulation of CD96 chemotherapy resistance

1. CD155 knockout regulated by CRISPR-CAS9

CRISPR direct is used to design the target sequence of CD155. In the present invention, the CD155 sequence of 5'-GTCACAGCTGACTTGGGCG-3' (SEQ ID NO. 5) is used as the target. Insert this sequence into the pCas9/gRNA vector to construct a sense oligonucleotide (SEQ ID NO.6): 5'ACACCGGTCACAGCTGACTTGGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT3' and an antisense oligonucleotide (SEQ ID NO.7): 3'TGTGGCCAGTGTCGACTGAACCCGCCAAAATCTCGATCTTTATCGTTCAATTTTCCGATCAGGCAA5', using water Dilute the oligonucleotide to 100 μM.

Prepare the annealing reaction system as follows to synthesize the fragment: 5 μl of sense oligonucleotide, 5 μl of antisense oligonucleotide, 100 mM NaCl (final concentration), Tris-Cl pH7.4 50 mM (final concentration), and add water to make up 50 μl. The synthesis was carried out with the program of 90°C for 4 minutes, 70°C for 10 minutes, 55°C for 10 minutes, 40°C for 10 minutes, and 25°C for 10 minutes.

2 μg pCas9/gRNA vector (inovogen) was digested with EcoRV. After digestion, use agarose gel to recover the linearized vector. The digested products were recovered using low melting point agarose gel and mixed with the annealed products of the above oligonucleotides, and then ligated with T4 DNA ligase overnight. Ligation reaction system: T4 DNA ligase 5U, EcoRV 5U, linearized vector 2μl, double-stranded oligonucleotide 1μl diluted 100 times, 10× ligase Buffer 1μl, 50% PEG40001μl, add water to make up 10μl, reaction conditions: 22°C 30min, 37℃15min. The ligated recombinant plasmid was transformed into competent DH5α bacteria (Takara Company). After screening positive clones in puromycin medium, single colonies were selected for expansion culture. Plasmids were extracted using the Omega kit and further identified by DNA sequencing and further transfected into MDA-MB-231 cells.

2. Effect of CD155 knockout tumor cells on chemotherapy

MDA-MB-231 breast cancer cells were treated with 0.02 μg/mL docetaxel alone or combined with 50 μg/mL CD96 neutralizing antibody for 24 hours after knocking out the expression of CD155 using CRISPR-CAS9 technology. Flow cytometry was used to detect the apoptosis of tumor cells (specific experimental steps are the same as before).

3. Changes in tumor cell chemotherapy after mutating CD96 and CD155 binding sites

The first Ig domain of CD96 is the main motif for binding to CD155, and the 75th tyrosine site is the most important amino acid for binding between the two, and is the main site for CD155 to activate CD96. Therefore, the applicant constructed a mutation site for the 75th tyrosine, and mutated the amino acid at this site to alanine (referred to as T75A). The sequence (SEQ ID NO. 8) is:

GCTGTCTATCATCCCCAAGCTGGCTTCTACTGTGCCTAT;

Reaction system for mutation: 10x pyrobest Buffer: 5ul, dNTP Mixture (10mM): 1ul, template DNA (5~50ng): 1ul, primer 1 (125ng): 1ul, primer 2 (125ng): 1ul, pyrobest DNA polymerase (TaKaRa )(5U/ul): 0.25ul, finally add sterile distilled water to 50ul. The total number of cycles is 15. Wash with sodium acetate and 75% ethanol, perform DpnI digestion (digestion at 30°C for 1 to 4 hours), and then terminate the reaction in a 65°C water bath for 15 minutes.

The mutated CD96 variant was inserted into the EcoRI/NotI site of the pcDNA3.1-6his plasmid, bacterial screening of mutants was performed with ampicillin, and large-scale plasmids were extracted and transfected into the indicated cells. TUNEL staining was used to observe the sensitivity of MCF-7 cells transfected with the mutant (these cells normally express low CD96) to neoadjuvant chemotherapy.

result:

1.1 CRISPR-CAS9 technology results knocked out the expression of CD155 in MDA-MB-231 breast cancer cells, indicating that the construction of CD155 sgRNA was successful (Figure 10).

1.2 After knocking out CD155, the sensitivity of MDA-MB-231 breast cancer cells to chemotherapy drugs was significantly enhanced, and the tumor killing capacity was the same as using CD96 neutralizing antibodies. However, CD96 neutralizing antibody combined with CD155 knockout cannot promote each other, indicating that the two pathways in regulating drug resistance are the same. The comprehensive function of CD96 in immune cells requires the action of the ligand CD155, so the CD155-CD96 signaling pathway is the activation combination for CD96 to exert drug resistance (Figure 11).

1.3 Mutating the 75th amino acid position of CD96 can significantly enhance the sensitivity of CD96-overexpressing MCF-7 cells to neoadjuvant chemotherapy drugs after inhibiting the binding of CD96 to CD155 (Figure 12).

The above results indicate that the interaction of CD155 is required for CD96 to regulate the drug resistance pathway of tumor cells.

in conclusion:

The neoadjuvant chemotherapy resistance marker of the present invention is specifically CD96. Through molecular biology research on clinical breast cancer tissue specimens, cohort follow-up data and in vitro cell models in his own work, the inventor found that CD96 itself is expressed on the surface of immune cells. It is highly expressed in breast cancer cells or cell lines in some patients and is positively correlated with poor prognosis after breast cancer chemotherapy. Further immunofluorescence and flow cytometry experiments showed that CD96 inhibits the sensitivity of breast cancer cells to neoadjuvant chemotherapy drugs. Knocking down the expression of CD96 ligand CD155 can have the same chemotherapeutic effect as inhibiting CD96, and mutating the binding site of CD96 and CD155 can significantly inhibit the drug resistance regulatory effect of CD96.

Based on this, we speculate that CD155 binds and activates CD96 to promote tumor cell resistance to neoadjuvant chemotherapy drug treatment. The discovery of this marker can, on the one hand, identify the relationship between the CD96 pathway in regulating tumor drug resistance through in-depth research, and provide scientific basis for new ways of targeting this pathway to treat breast cancer. On the other hand, it can also provide tips for people who are resistant to neoadjuvant chemotherapy, and play a role in personalized treatment and precision treatment in breast cancer treatment or other cancer treatments.

The above are only preferred embodiments of the present invention and do not limit the present invention in any form. Therefore, any simple modifications to the above embodiments may be made based on the technical essence of the present invention without departing from the technical content of the present invention. , equivalent changes and modifications, all still fall within the scope of the technical solution of the present invention.

Claims (1)

1. The application of CD96 inhibitors in the preparation of products that improve the sensitivity of neoadjuvant chemotherapy for breast cancer is characterized by: The CD96 inhibitor is selected from the group consisting of CD96 neutralizing antibodies, shRNA designed based on CD96, and sgRNA designed based on the CD96 upstream regulatory gene CD155; The shRNA designed based on CD96 is shown in SEQ ID NO.3 or SEQ ID NO.4; The sgRNA designed based on the CD96 upstream regulatory gene CD155 is shown in SEQ ID NO. 6-7.