CN116286831B - Nucleic acid aptamer of targeted EGFR T790M mutant and application thereof in preparation of lung cancer treatment drugs - Google Patents

Abstract

The invention discloses a nucleic acid aptamer targeting EGFR T790M mutant and application thereof in preparing a medicine for treating lung cancer. The invention successfully screens and obtains the nucleic acid aptamer T1, T2, T3, T4, T5, T6 and T7 which can combine EGFR T790M mutant with high affinity through the SELEX technology, and proves that the nucleic acid aptamer has the functions of inhibiting proliferation, migration and invasion of lung cancer H1975 cells expressing the mutation, and the administration of the nucleic acid aptamer to a lung cancer subcutaneous tumor model nude mouse has the function of inhibiting proliferation of tumor bodies. Provides a new method and thinking for developing new specific lung cancer targeted drugs.

Description

Nucleic acid aptamer targeting EGFR T790M mutant and its use in preparing drugs for treating lung cancer Application in things

Technical areas:

The invention belongs to the field of biomedicine, and specifically relates to a nucleic acid aptamer targeting the EGFR T790M mutant and its application in preparing drugs for treating lung cancer.

Background technique:

Lung cancer is the most common malignant tumor. It has the characteristics of strong insidiousness, high mortality risk, and heavy burden of morbidity. Its incidence and mortality are still on the rise. Lung cancer is divided into two pathological types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). About 80% to 85% of lung cancer patients are NSCLC. Since lung cancer has no obvious diagnostic features in its early stages, most lung cancer patients are already in the mid-to-late stage when diagnosed. Although commonly used clinical treatments for lung cancer continue to mature, many patients still face risks such as postoperative recurrence, tumor metastasis, and poor prognosis. Clinically, lung cancer treatment is mainly based on platinum-based chemotherapy drugs. Surgery is the first choice for early-stage NSCLC. However, many patients have a high risk of postoperative recurrence and require adjuvant radiotherapy and chemotherapy in the later stage. Studies have shown that the 5-year survival rate after surgery for stage III NSCLC is less than 25%. Radical radiotherapy or stereotactic ablative radiotherapy (SABR) are also optional treatments. Treatment options for patients with advanced NSCLC include surgery plus postoperative radiotherapy. Chemotherapy or palliative care. In the past 20 years, our understanding of the mechanisms of lung cancer development has continued to improve. Targeted therapeutic drugs for lung cancer that are widely used in clinical practice such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR) -TKI) and immunotherapy have improved the survival of patients with advanced non-small cell lung cancer (NSCLC) to a certain extent.

Epidermal growth factor receptor (EGFR) belongs to the tyrosine kinase receptor family, also known as HER1 (ERBB1), and is the expression product of the proto-oncogene. Members of this family also include HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB3). EGFR consists of 1186 amino acid residues and consists of four parts: extracellular ligand domain, transmembrane domain, intracellular tyrosine kinase domain and carboxyl terminal regulatory region. The extracellular ligand-binding region of EGFR can bind to corresponding ligands (such as EGF or TNF-α, etc.) to form homodimers or heterodimers. Phosphorylation with the participation of ATP triggers autophosphorylation of specific tyrosine residues in the cytoplasmic regulatory domain, thereby activating downstream signaling pathways. When the EGFR signaling pathway is activated, it can participate in regulating physiological activities such as cell proliferation, survival, invasion, and blood vessels. Therefore, when EGFR is mutated, it can activate downstream signaling pathways and promote cell proliferation and differentiation and the metastasis of malignant tumors.

EGFR mutation is the most common driver gene of advanced lung adenocarcinoma. EGFR-TKI is one of the main methods of treating advanced NSCLC. The first and second generation EGFR-TKI drugs include gefitinib, erlotinib and afatinib. etc., targeted EGFR mutations include deletion of exon 19 and L858R point mutation of exon 21. Its mechanism of action is to competitively act on ATP and intracellular tyrosine kinase domain sites, preventing ATP from participating in EGFR phosphorylation, and can significantly prolong the average progression-free survival (PFS) of patients with EGFR-activated tumors. ). However, secondary resistance caused by secondary mutations in EGFR greatly affects the efficacy of the first and second generation EGFR-TKIs. Studies have found that 50% of EGFR-TKI-resistant patients have a point mutation at nucleotide 2369 of EGFR exon 20, also known as the T790M mutation. In the T790M mutation, methionine (M) replaces the original threonine (T), resulting in changes in the spatial structure of EGFR, hindering the normal binding of EGFR to first- and second-generation EGFR-TKIs, and increasing the interaction between EGFR and ATP. Binding affinity causes a series of cascade reactions, reactivates downstream pathways, and inhibits cell apoptosis. Therefore, the T790M mutation is a hot topic in research on early diagnosis of lung cancer, targeted therapeutic drugs, drug resistance detection and other related research. It is of important clinical significance to continue to study new drugs based on exploring the treatment of lung cancer targeting the T790M mutation.

Targeted therapy of tumors requires ligands that specifically bind to cancer cells. In addition to inhibitors and antibodies, new tumor-targeting ligands such as aptamers have emerged in the past few decades, which can be used in tumor diagnosis and treatment applications. targeting molecules. Aptamers are also called nucleic acid aptamers, aptamers, and ligands. They usually use random nucleotide sequences with a length of 20-100nt as the initial library. They are a ligand system evolution technology (systematic evolution) through in vitro exponential enrichment. Single-stranded DNA or RNA molecules obtained by screening of ligands by exponential enrichment (SELEX). In the early 1990s, Tuerk and Gold ^[23,24] first proposed the concept of aptamers. Although aptamers have a small molecular weight, they can be folded into a variety of complex three-dimensional spatial structures to interact closely with target molecules, and then bind to the corresponding target molecules with high affinity and specificity. They are considered by the industry to have antibody properties. At the same time, compared with antibodies, aptamers still have many characteristics: (1) small molecular weight and easy synthesis: aptamers are generally less than 100nt, obtained through in vitro screening, mature technology, short time-consuming, and low cost; ( 2) Wide range of targets: including metal ions, nucleic acids, peptides, growth factors, neurotransmitters, cells, viruses, bacteria, etc.; (3) Good stability: aptamers have a long half-life, are not sensitive to heat, and can be denatured by chemical factors The active conformation can still be restored under appropriate conditions, so it can be stored and transported stably for a long time, and the variation between batches is small; (4) Easy to modify: In order to realize the carrier function of the aptamer, prevent nuclease degradation and improve stability, For further application in clinical diagnosis and treatment, special sites of aptamers are usually modified, such as combining radioactive isotopes, avidins, enzymes, nanoparticles, etc.; (5) Low toxicity, low immunogenicity, etc.

SELEX technology is an in vitro screening method based on the natural evolutionary mechanism of organisms. The basic principle and process is to select appropriate target molecules according to the purpose of the experiment, design and construct a random nucleotide library as the initial library, specifically combine the nucleotide sequence with the target through different methods, remove non-specific nucleic acid sequences, and combine the nucleotide sequences that specifically bind to the target molecule. The target nucleic acid fragments are amplified and enriched by PCR and then re-entered into the next round of screening. Through multiple rounds of repeated screening, an aptamer that binds to the specific fragment with high specificity and high affinity is finally obtained. After more than 20 years of development, scientific researchers have continuously improved SELEX technology in order to improve screening efficiency, binding affinity and stability. The current new SELEX technologies include capillary electrophoresis SELEX technology (Capillary Electrophoresis SELEX), microfluidic chip SELEX technology (Microfluidics SELEX), cell-SELEX technology and magnetic beads SELEX technology (Magnetic beads SELEX). Among them, magnetic bead SELEX technology is one of the commonly used new SELEX technologies. It uses the characteristics of large surface area, small particle size, easy modification and separation of magnetic beads to bind the target molecules to the surface of the magnetic beads, and then incubates with the nucleotide library and screens to obtain Specific aptamer sequences. This process is simple to operate and can quickly separate target sequences, greatly reducing screening time and cost and improving screening efficiency.

With the maturity and advancement of SELEX technology, many aptamers have been screened out that specifically bind target tumor markers and tumor cells, and are used in targeted cancer therapy, drug delivery, molecular imaging and other aspects. Because aptamers have the characteristics of small molecular weight, high targeting ability, and high tissue permeability, they are ideal molecular imaging probes and targeted therapeutic molecules. As a nucleic acid molecule, it has good application prospects in tumor diagnosis and treatment. In 2004, the first aptamer drug, pegaptanib sodium (Macugen), was approved by the U.S. Food and Drug Administration (FDA) and targeted the vascular endothelial growth factor receptor (VEGF) for the treatment of age-related macular degeneration. Degenerative diseases. At the same time, it has been verified that the aptamer can be used as a nucleic acid therapeutic molecule that binds to the target with high specificity and high affinity, and plays a role in inhibiting overexpressed or mutated related target proteins in tumors.

Contents of the invention

The first object of the present invention is to provide a nucleic acid aptamer that specifically binds to the EGFR T790M mutant.

The nucleic acid aptamer of the present invention that specifically binds to the EGFR T790M mutant protein has any one of the following sequences:

T1:

5'-ATCCAGAGTGACGCAGCATTTTGACGCTTTATCCTTTTCTTATGGCGGGATAGTTTCGTGGACACGGTGGCTTAGT-3'; (as shown in SEQ ID NO.1)

T2:

5'-ATCCAGAGTGACGCAGCATTTTGACGCTTTATCCTTTTCTTATGGTGGGATAGTTTCGTGGACACGGTGGCTTAGT-3'; (as shown in SEQ ID NO. 2)

T3:

5'-ATCCAGAGTGACGCAGCATAATAGGCCATGGGTGGTACTGTGGCAATCCCCAGGCTCTTGGACACGGTGGCTTAGT-3'; (as shown in SEQ ID NO.3)

T4:

5'-ATCCAGAGTGACGCAGCAAACCAACGGACTGGTCATTGGTCACGTCCGGAAAGGCTAATGGACACGGTGGCTTAGT-3'; (as shown in SEQ ID NO.4)

T5:

5'-ATCCAGAGTGACGCAGCACTTGAAAGGTGGGTGAGCTGAAATAAATTCATGCTTTAGCTGGACACGGTGGCTTAGT-3'; (as shown in SEQ ID NO.5)

T6:

5'-ATCCAGAGTGACGCAGCAGAGGCCATGGAGCAAGGGTATACCGTTTACTTCTTGTGTGGACACGGTGGCTTAGT-3'; (as shown in SEQ ID NO. 6)

T7:

5'-ATCCAGAGTGACGCAGCAGTCTAGGGCATCCGTGCCGCTATATGAACAGGTTATATGTGGACACGGTGGCTTAGT-3' (shown in SEQ ID NO. 7).

Preferably, the nucleic acid aptamer is T2.

The second object of the present invention is to provide the application of the above-mentioned nucleic acid aptamer that specifically binds to the EGFR T790M mutation in the preparation of drugs for the treatment of lung cancer.

Preferably, it is the application of nucleic acid aptamers in drugs that inhibit the proliferation, migration and invasion of lung cancer H1975 cells expressing EGFR T790M mutant protein.

Preferably, it is the application of nucleic acid aptamers in drugs that inhibit tumor proliferation of subcutaneous lung cancer tumors.

The third object of the present invention is to provide a drug for treating lung cancer, which contains the above-mentioned nucleic acid aptamer as an active ingredient.

Preferably, the nucleic acid aptamer contains pharmaceutically acceptable excipients.

The present invention uses magnetic bead SELEX technology and uses T790M mutant polypeptide as the target molecule. The carboxyl and amino groups on the surface of the magnetic beads can be coupled to the target polypeptide after activation, and the random oligonucleotide library synthesized in vitro is incubated and amplified with the target. and separation, and finally screen to obtain highly enriched DNA nucleic acid sequences that specifically bind to the target. During each round of screening, it is necessary to repeatedly explore experimental conditions and explore the optimal cycle number and optimal reaction system for asymmetric PCR amplification to obtain DNA sequences with high purity and single bands. Otherwise, the ssDNA finally obtained through the magnetic bead separation method is easily mixed with contaminants, causing off-target phenomena and cannot be used as a secondary library to enter the next round of screening. At the same time, counter-screening polypeptides were introduced from the third round of screening, and the counter-screening step was added to elute away the ssDNA bound to the counter-screening polypeptides, leaving the ssDNA not bound to the counter-screening polypeptides as a sub-library for the next round of screening, further improving the Screening efficiency. After 9 rounds of screening, the target band with a molecular weight of 76nt, high enrichment and high purity was obtained. The target band is PCR amplified and gel purified before high-throughput sequencing. Compared with traditional cloning sequencing, high-throughput sequencing has a greater detection range for screening products, and the detection capacity reaches more than 2G, which can prevent the missed detection of high affinity. Sexual aptamer.

The high-throughput sequencing results showed that the screening product was of high purity and contained multiple highly enriched DNA sequences. Through homology sequence comparison, seven highly enriched and highly homologous ssDNA sequences of 76 nt were obtained, which were named adapters. Body T1, T2, T3, T4, T5, T6, T7. Secondary structure prediction showed that they all have similar stem-loop structures, suggesting that they may serve as the main sites for aptamers to specifically recognize mutant fragments.

Flow cytometry can measure the fluorescence intensity of fluorescently labeled aptamers after binding to target cells. Lung cancer H1975 cells naturally express the EGFR T790M mutation. Flow cytometry is used to detect the binding of the screened aptamers to lung cancer H1975 cells. It can be judged whether the aptamer can bind the target with high specificity. Through flow cytometry analysis, it was found that the aptamer was highly bound to H1975 cells, and the fluorescence peaks were shifted to the right. The aptamer T2 had the highest binding ability to H1975 cells, with a relative binding rate higher than 85%. There was no difference in the binding between aptamer and A431 cells expressing EGFR ^WT between the blank group and the random control Scr group. At the same time, different concentration gradients of FAM-T2 and random control aptamer FAM-Scr were incubated with lung cancer H1975 cells. Flow cytometry was used to detect the affinity of aptamer T2 to H1975 cells. The experimental results showed that the concentration of aptamer T2 is proportional to the fluorescence intensity. The K _d value of aptamer T2 is 30.54±5.859nM. The random control aptamer Scr The K _d value is 81.52±22.29nM. Aptamer T2 has high affinity with H1975 cells. In summary, it shows that the aptamer T2 obtained through screening can bind to H1975 cells expressing the T790M mutation with high affinity and specificity.

The effect of aptamer T2 on the proliferation, migration and invasion ability of H1975 cells was explored at the cellular level. H1975 cells expressing T790M mutation were used as target cells, and skin cancer A431 cells expressing EGFR ^WT were used as control cells. The CCK-8 method was used to verify whether aptamer T2 can specifically affect the survival rate of H1975 cells in vitro and inhibit the proliferation of lung cancer H1975 cells. At the same time, as the dosage concentration and time increase, the in vitro survival rate of H1975 cells decreases; the effect of aptamer T2 on the proliferation ability of H1975 cells was further explored through the EdU method. The results showed that aptamer T2 can significantly inhibit lung cancer H1975 cells. proliferation ability. A scratch experiment was conducted to explore the effect of aptamer T2 on H1975 cells after scratching. The relative healing area was significantly lower than that of the blank group and the control group, indicating that aptamer T2 can inhibit the migration ability of H1975 cells. At the same time, the Transwell experiment was conducted to explore the effect of aptamer T2 on the migration and invasion ability of H1975 cells. The experimental results showed that aptamer T2 can specifically inhibit the migration and invasion ability of H1975 cells. At the same time, in the cell function level verification experiment, the third-generation EGFR-TKI osimertinib (AZD9291) was introduced as a positive control. The experimental results found that osimertinib (AZD9291) acted on H1975 cells and A431 cells, both with obvious effects. The effect of inhibiting cell proliferation, migration and invasion is stronger, and the effect of drugs on inhibiting cell activity is stronger. This shows that AZD9291 has a significant anti-tumor effect on both cell lines expressing EGFR ^WT and EGFR T790M mutation, while aptamer T2 only has an effect on lung cancer cell lines expressing T790M mutation.

At the molecular level, the autophosphorylation of EGFR can continuously activate downstream signaling pathways. For example, EGFR dimerization stimulates Ras protein, which can further activate the AKT signaling pathway, which can continuously stimulate cell proliferation and growth, causing the occurrence and development of tumors. This project found through Western Blot experiments that after aptamer T2 treated lung cancer H1975 cells, the phosphorylation level of EGFR was reduced while the total protein level remained unchanged. The phosphorylation level of the downstream AKT protein was also reduced. Therefore, it was verified that aptamer T2 may pass Inhibiting the phosphorylation levels of EGFR and AKT inhibits cell proliferation. A subcutaneous lung cancer nude mouse model was successfully established at the animal level, and the in vivo tumor inhibitory effect of the aptamer T2 targeting the T790M mutation was explored through intratumoral injection. The experimental results show that the aptamer T2 group has a certain inhibitory effect on tumor growth than the control group. At the same time, the survival time of nude mice after intratumoral injection of aptamer T2 is longer, and their average survival time is increased than that of the control group (P<0.05 ). It provides new ideas for targeted treatment of lung cancer.

Picture description:

Figure 1 is the polyacrylamide natural gel and denaturing gel electrophoresis diagram;

A: 10% native gel electrophoresis identifies PCR products at different cycle numbers (lane 1: pUC18 DNA Maker; 2-5: PCR products when amplification cycle numbers are 6, 8, 10, and 12);

B: 8% denaturing gel identification and screening of target ssDNA (lane 1: pUC18 DNA Maker; 2: original library; 3-4: ssDNA separated by magnetic beads),

Figure 2 shows the secondary structure of the aptamer sequence calculated by Mfold software.

Figure 3 shows the flow cytometry detection of the binding specificity of aptamers and H1975 cells.

A: Flow cytometry detects the binding of aptamer FAM-T1-T7 and control aptamer FAM-Scr to H1975 cells and A431 cells;

B: Analysis of the absolute binding rate of aptamer T2 and Scr binding to H1975 cells and A431 cells, ***P<0.001.

Figure 4 shows the affinity between the aptamer and H1975 cells detected by flow cytometry;

A: Fitting curve of aptamer Scr dissociation constant K _d ;

B: Fitting curve of aptamer T2 dissociation constant K _d .

Figure 5 CCK8 detects the relative cell survival rates of aptamers T2, Scr, and AZD9591 acting on A431 cells and H1975 cells respectively.

Figure 6 shows the effects of aptamers T2, Scr and AZD9291 on the proliferation ability of H1975 cells;

A: Aptamer T2 and AZD9291 significantly inhibit the proliferation of H1975 cells;

B: Analysis of relative proliferation rates of different treatment groups. **P<0.01, ***P<0.001.

Figure 7 shows the scratch experiment to detect the effect of aptamer T2 on the migration of H1975 and A431 cells;

A: Lung cancer H1975 cells treated with scratches by different groups under the microscope;

B: Analysis of the relative scratch healing area rate of different groups on H1975 cells, **P<0.01, ***P<0.001;

C: A431 cells after scratch treatment by different groups under the microscope;

D: Analysis of relative scratch healing area rates of different groups on A431 cells, *P<0.05.

Figure 8 is a Transwell cell migration experiment to detect the effects of T2, Scr, and AZD9291 on cell migration ability after 24 hours of treatment;

A: Representative graph of cell migration experiment represents the difference in cell migration ability in each experimental group;

B: Statistical values Compare the cell migration numbers of T2 and Scr. Each independent experiment is repeated three times. There is a significant difference in the cell migration ability between the H1975 cell T2-treated group and the Scr-treated group, ***P<0.001.

Figure 9 is a Transwell experiment to detect the effect of aptamer T2 on the invasion ability of H1975 cells;

A: Cells that invaded in each experimental group;

B: Statistical values compare the number of cells that invaded in the T2 and Scr groups. Each independent experiment was repeated three times. There was a significant difference in the cell invasion ability between the H1975 cell T2-treated group and the Scr-treated group, ***P<0.001.

Figure 10 is a study on the mechanism of aptamer T2 inhibiting the phosphorylation levels of EGFR and AKT;

A: Comparing the blank group and the control group, aptamer T2 can significantly reduce the phosphorylation level of EGFR protein, **P<0.01, but does not affect the expression of total EGFR protein;

B: Comparing the blank group and the control group, aptamer T2 can reduce the phosphorylation level of AKT protein, **P<0.01.

Figure 11 is to explore the in vivo tumor inhibitory effect of aptamer T2 in nude mice of lung cancer model;

A: Tumor tissue stripped from the blank group (NaCl), control group (Scr) and experimental group (T2);

B: Statistics of tumor volume in each group, **P<0.01;

C: Survival curves of blank group (NaCl), control group (Scr) and experimental group (T2);

D: Statistics of average survival time of each group, ***P<0.001.

Detailed ways:

The following examples further illustrate the present invention, rather than limiting the present invention.

Example 1: Using magnetic bead SELEX method to screen aptamers targeting EGFR T790M mutation

Experiment 1. Library construction, primer synthesis and target peptide synthesis

1. Select an appropriate random ssDNA library based on the screening object. A random ssDNA library with a length of 76nt was synthesized by Invitrogen Company. It has 18 sequences at each end as fixed sequences and 40 random sequence fragments in the middle. The primer sequences were determined and synthesized based on the sequences at both ends of the library and were labeled with -FAM and -Biotin tags respectively. The sequences are as follows.

GN original library: 5'-ATCCAGAGTGACGCAGCA (N40)GGACACGGTGGCTTAGT-3' (N=A, T, C, G)

Primer 1 sequence: FAM-GN1: 5'-ATCCAGAGTGACGCAGCA-3'

Primer 2 sequence: Biotin-GN2: 5'-ACTAAGCCACCGTGTCCA-3'

Randomized control Scr:

5'-ATCCAGAGTGACGCAGCACTTTAAGACTTCTTAGTGTTGCTGTTTGTGTATTTCTCGGTGGACACGGTGGCTTAGT-3'

2. Query the amino acid sequence of EGFR protein through NCBI (NCBI accession number NM_005228.5), find the 790th amino acid residue in the amino acid sequence of EGFR protein, and select a polypeptide with a length of 21 amino acid residues centered on this site. Fragments, called EGFR polypeptides, serve as counter-screening peptides for aptamer screening.

A 21-amino acid residue sequence in which threonine (T) at the same position was replaced with methionine (M) was selected and named EGFR T790M polypeptide. It was used as the target polypeptide for aptamer screening and was developed by Shanghai Qiangyao Biotech. Synthesized, the sequence is as follows.

EGFR T790M peptide: ICLTSTVQLIMQLMPFGCLLD

EGFR peptide: ICLTSTVQLITQLMPFGCLLD

Experiment 2: Screening of nucleic acid aptamers targeting EGFR T790M mutant using magnetic bead SELEX technology

1. The first two rounds of target peptide screening: activate the -COOH and -NH2 groups of magnetic beads: mix the SLE-embedded silica magnetic microspheres with -COOH and -NH2 groups in a oscillator, and take the Mix 200 μL of magnetic beads in an EP tube, magnetically separate the beads using a magnetic stand, and discard the supernatant. Add 1.0 mL PBS to resuspend the pellet, mix well, remove the supernatant by magnetic separation, and repeat washing three times. Add 200 μL of 40mM EDC solution to resuspend the pellet to activate the -COOH and -NH3 groups of the SLE-embedded silica magnetic microspheres; bind to the target peptide: take an appropriate amount of the target peptide T790M peptide and add it to the activated SLE-embedded silica. Incubate in magnetic microspheres for 2 hours on a shaking table at 4°C. After 2 hours, magnetically separate the magnetic beads by a magnetic stand, discard the supernatant, resuspend the pellet in 1.0 mL PBS, magnetically separate and remove the supernatant, and repeat washing three times; prepare the ssDNA library: select the synthesized ssDNA (GN) library in the first round of screening ( The sample input in the subsequent screening is the ssDNA obtained in the previous round of screening), add 200 μL PBS to dilute, and add a certain concentration of yeast tRNA and salmon sperm DNA. Heat the sample in a metal bath at 95°C for 5 minutes, cool it on ice for 5 minutes, add 500 μL of binding buffer and let it stand at room temperature for more than 30 minutes; obtain the ssDNA bound to the EGFR T790M polypeptide: mix the ssDNA library with the SLE magnetic beads bound to the target molecule T790M polypeptide, and mix by pipetting Homogenize for more than 5 minutes, mix on a 37°C shaker and incubate for 30 minutes, then magnetically separate and discard the supernatant. Take 1.0mL of binding buffer to resuspend the pellet, magnetically separate to remove the supernatant, repeat the above steps twice, and finally resuspend with 300μL of binding buffer. precipitation. Heat the sample in a metal bath at 95°C for 10 minutes, mix by pipetting for 2-3 minutes, separate on a magnetic stand and collect the supernatant to obtain ssDNA bound to the EGFR T790M polypeptide, and use this as a template for asymmetric PCR amplification.

2. After the third round, introduce EGFR polypeptide as a counter-screening polypeptide: follow the same operation as step 1 to activate SLE-embedded silica magnetic microspheres and combine EGFR polypeptide with SLE magnetic beads; repeat step 1 to obtain EGFR T790M polypeptide-bound ssDNA, add it to the SLE magnetic beads bound to the EGFR polypeptide, mix by pipetting for more than 5 minutes, mix on a shaker at 37°C and incubate for 30 minutes, then magnetically separate the magnetic beads through a magnetic stand, discard the magnetic beads, and recover the supernatant, which means it has not been screened with reverse screening. Peptide-bound ssDNA. Heat the sample in a metal bath at 95°C for 10 minutes, pipet and mix for 2-3 minutes, magnetically separate the remaining magnetic beads through a magnetic stand, collect the supernatant, and use this as a template for the next step of PCR amplification;

There are 9 rounds of cyclic screening in this way. The input amount during each round of screening is as follows:

Table 1 The input amount of each component in each round of aptamer screening

3. Explore the optimal number of cycles for PCR amplification: Prepare the PCR reaction system according to Table 2; PCR reaction conditions: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, and the number of amplification cycles is 6 +4X cycles (X=1, 2, 3, 4...), final extension at 72°C for 5 minutes.

Table 2 PCR reaction system

4. Identification of 10% polyacrylamide natural gel electrophoresis: Preparation of polyacrylamide natural gel: Select a 10cm × 8cm glass plate, clean it, dry it, assemble the gel mold, and use ddH ₂ O to check whether there is leakage, and confirm that there is no leakage. Discard the ddH ₂ O after leakage occurs. Take a 50.0mL centrifuge tube, prepare gel according to the formula, and shake gently in a shaker. Slowly pour the prepared gel into the gap in the middle of the glass plate, insert a comb of matching size, and be careful not to leave air bubbles. Let stand at room temperature for more than 30 minutes until the gel solidifies; load the sample: Assemble the electrophoresis equipment, put the glass plate with gel into the electrophoresis tank, add 1×TBE electrophoresis solution until the sample hole is covered, and carefully pull out the comb vertically. Mix the amplified DNA sample and 6× loading buffer at a ratio of 5:1. Use a micropipette gun to add the DNA sample and 5 μL pUC 18DNA/Msp Maker to the loading hole; electrophoresis: fill the electrophoresis tank with 1×TBE electrophoresis solution, cover the electrophoresis cover, connect the power supply correctly, and perform electrophoresis under 140V constant voltage conditions; staining and imaging: Remove the glass plate, slowly place the gel horizontally in the staining tank, add 20mL ethidium bromide staining solution (0.5μg/mL ethidium bromide, prepared with 1×TBE solution) for 20 minutes, then remove the gel and place it in the gel Imaged in the imager.

Table 3 Polyacrylamide natural gum formulation table

5. Separate ssDNA by streptavidin magnetic bead separation method: Wash the streptavidin magnetic beads: Take out 200 μL streptavidin magnetic beads, oscillate and mix in a oscillator, add 1 mL of PBS, oscillate and mix for 1 min. Centrifuge at 800 rpm/min for 3 min at room temperature, discard the supernatant, and repeat washing with PBS three times. Finally, add 800 μL PBS to resuspend; combine streptavidin magnetic beads with dsDNA: take the dsDNA amplified and identified in step 4, and mix on a shaker at 4°C for 1 hour. Centrifuge at room temperature 800rpm/min for 3 minutes, discard the supernatant, add 1.0 mL PBS to resuspend the pellet, mix by pipetting for 1 minute, centrifuge at room temperature 800rpm/min for 3 minutes, discard the supernatant, repeat washing with PBS 3 times, centrifuge at room temperature 8000rpm/min for 5 minutes, try to Remove the supernatant; alkaline lysis and neutralization: add 500 μL 0.2M NaOH to resuspend the pellet, mix by pipetting for 15 min, and then centrifuge at 8000 rpm/min for 5 min to collect the supernatant. Add 1/10 volume of 2M acetic acid to the supernatant; purify ssDNA: measure the total volume of supernatant ssDNA, and add 1/10 times the volume of 3mol/L NaAC, 1/100 times the volume of 1mol/L MgCl2, and 2.5 according to the sample volume. twice the volume of absolute ethanol, place the sample in a -80°C refrigerator overnight (>24h), then thaw on ice, centrifuge at 4°C, 14,000rpm/min for 30 minutes, remove the supernatant, add 350 μL of 70% ethanol to dissolve the precipitate, 4 Centrifuge at 14,000 rpm/min for 30 minutes, remove the supernatant, open the lid of the EP tube, and leave it at room temperature for more than 3 hours until the sample is dry. Then take an appropriate amount of ddH2O to dissolve the sample, use a UV spectrophotometer to detect the sample concentration, and store it in a -20°C refrigerator. As a sub-library for the next round of screening, another 5 μL sample was taken for identification by denaturing gel electrophoresis.

6. Identification of 7M urea 8% denaturing gel electrophoresis: Preparation of 7M urea 8% denaturing gel: The preparation operation is consistent with the step 4 of polyacrylamide natural gel. The formula is as shown in Table 4. Assemble the electrophoresis equipment and add 1× to the electrophoresis tank. TBE electrophoresis solution, pull out the comb vertically. Because the loading hole of the denaturing gel is prone to urea precipitation, a 200 μL pipette is required to punch the hole; mix the separated ssDNA sample and 2× denaturing loading buffer at a ratio of 1:1; use a metal bath 95 to mix the sample Heat for 5 minutes at ℃ and then incubate on ice for 5 minutes. Use a micropipette gun to add the ssDNA sample and 5 μL pUC 18DNA/Msp Maker to the loading hole; fill the electrophoresis tank with 1×TBE electrophoresis solution, cover the electrophoresis cover, and connect the power supply correctly according to the positive and negative poles, 100V Electrophoresis under constant voltage conditions, until the sample reaches the bottom of the gel, cut off the power supply; remove the glass plate, place the gel in a staining tank, and add 20.0 mL of ethidium bromide staining solution (0.5 μg/mL ethidium bromide, 1 × TBE solution) After staining for 20 minutes, take out the gel and image it in a gel imager.

Table 4 Polyacrylamide denaturing glue formulation table

7. Sequencing and structural analysis: PCR amplify the ssDNA obtained in the last round of screening to obtain dsDNA, identify the target band through polyacrylamide native gel electrophoresis, cut out the target gel with a fragment length of 76 bp under UV light, and use Absorb excess water with filter paper, put it into a 1.5mL EP tube, and weigh the gel (minus the weight of the empty tube). Use DNA purification kit from Nanjing Novizan Vazyme Company ( Gel DNA Extraction mini kit, DC301) to obtain purified DNA; send it to Guangzhou Kidio Company for high-throughput sequencing (sequencing volume >2G); use Mfold software to calculate and screen the high-volume repetitive fragments obtained by high-throughput sequencing to obtain suitable Secondary structure and minimum free energy of the ligand.

Experimental results:

1. Identification of ssDNA for magnetic bead SELEX screening: Target EGFR T790M mutation through magnetic bead SELEX technology. During each round of screening, a nucleotide library that specifically binds to the target polypeptide is obtained, amplified and enriched by PCR to obtain dsDNA, and then FAM-ssDNA is separated by magnetic beads and used as a sub-library for the next round of screening. At the same time, anti-screening peptides were introduced in the third round to wash out ssDNA that was not bound to the anti-screening peptides, further improving screening efficiency. After 9 rounds of screening, highly enriched aptamers with high target binding were obtained. In each round of screening, the optimal amplification cycle number needs to be explored through asymmetric PCR amplification, and the PCR products at different cycle numbers must be identified through 10% polyacrylamide native gel electrophoresis. When the PCR amplification cycle number is 8, the band is the clearest and there are no contamination bands. When the cycle number is further increased, the band becomes more diffuse and contamination bands appear (Figure 1A). The final product was identified and screened through an 8% denaturing gel. As shown in Figure 1B, the target band was obtained with high purity, a single band, and a molecular weight of 76 nt.

2. Aptamer sequence analysis and secondary structure prediction: The aptamers obtained through screening were amplified by PCR, gel-cut and purified, and then subjected to high-throughput sequencing. It was determined that the screening products had multiple highly enriched DNA sequences. Through homology sequence comparison, seven aptamer sequences with a length of 76 nt and high homology were obtained and named T1-T7. The sequences are shown in Table 2. Use Mfold software to calculate the secondary structure and lowest free energy of the aptamer sequence. As shown in Figure 2, the seven sequences screened have similar stem-loop structures and have lower free energy, indicating that the aptamer structure obtained by screening is stable. , and the stem-loop secondary structure may be necessary for the aptamer to function.

Table 5 Aptamer sequences obtained by screening

3. Detect the binding specificity of the aptamer to H1975 cells by flow cytometry: Based on the 7 nucleic acid aptamers named T1-T7 obtained from SELEX, synthesize an aptamer sequence with a FAM label at the 5' end and random Control aptamer FAM-Scr. The cells were incubated with lung cancer H1975 cells expressing EGFR T790M mutation and skin cancer A431 cells expressing EGFR ^WT respectively. H1975 cells and A431 cells without aptamer were used as blank controls in each group. Flow cytometry was used to detect the binding of aptamers to H1975 cells and A431 cells. The test results showed that compared with the random control Scr, the fluorescence peaks of the seven aptamer sequences were all shifted to the right. The seven aptamer sequences screened have strong binding ability to H1975 cells expressing the target polypeptide. The fluorescence signals of the aptamer sequence and negative control A431 cells were basically the same as those of the blank group and the random sequence Scr group, and there was almost no change in the fluorescence peak (Figure 3A). This shows that the screened aptamer can specifically recognize lung cancer H1975 cells expressing the T790M mutation, and aptamer T2 has a better recognition effect on H1975 cells, with a binding rate greater than 85%, ***P<0.001. The binding rate of aptamer T2 to A431 cells was not high (Figure 3B). Therefore, the aptamer T2 can be obtained and can bind to H1975 cells with high specificity.

4. Detect the affinity of the aptamer and H1975 cells by flow cytometry: According to the results of 3, it was found that the aptamer T2 can highly specifically bind to H1975 cells. The corresponding concentration gradient FAM-T2 and the random control aptamer FAM-Scr were combined with Incubation of lung cancer H1975 cells expressing the EGFRT790M mutation. Flow cytometry was used to detect the fluorescence intensity of the aptamer combined with H1975 cells. The experimental results showed that the aptamer concentration is proportional to the fluorescence intensity. Nonlinear fitting was performed through GraphPad Prism9.0 to obtain the corresponding dissociation constant. The constant K _d represents the affinity of the aptamer to bind to lung cancer H1975 cells. The smaller the K _d , the higher the affinity of the aptamer to the lung cancer H1975 cells. As shown in Figure 4, the K _d value of aptamer T2 is 30.54± 5.859nM, the K _d value of the random control aptamer is 81.52±22.29nM. This shows that compared with the control group, aptamer T2 has higher affinity with H1975 cells.

Experiment 3: CCK-8 method to explore the effect of aptamer T2 on the proliferation of lung cancer H1975 cells

It was identified that the aptamer T2 has the highest binding degree to lung cancer H1975 cells. The aptamer T2 required for the experiment and the random control aptamer Scr were synthesized by Thermo Fisher; when H1975 cells and A431 cells are in the logarithmic growth phase, Discard the original culture medium, add PBS and wash once, then add 0.25% trypsin for digestion. When the cells fall off the culture wall, add complete culture medium to terminate the digestion; collect the cells into a 15mL centrifuge tube, centrifuge at 900rpm/min at room temperature for 5 minutes and then discard. Supernatant, add complete culture medium to resuspend the cells, inoculate the cell suspension evenly into two 96-well plates, control the cell density at 5,000 cells per well, and place them in a 37°C, 5% _CO2 cell culture incubator; The cells in the 96-well plate were randomly divided into 4 groups: blank group (no treatment, only basal medium 1640 and DMEM were added), experimental group (100, 200nM concentration of aptamer T2 treated for 24h, 48h), negative control group (100, 200nM aptamer Scr treated for 24h, 48h), positive control group (25, 50nM AZD9291 treated for 24h, 48h); after the cells were cultured overnight, discard the original culture medium of the 96-well plate, wash it with PBS and add The corresponding concentrations of aptamers T2, Scr and AZD9291 were prepared from the basal medium, and 1640 and DMEM basal medium were added to the blank group respectively, and 5 parallel wells were set in each group. Place the 96-well plate in a 37°C, 5% _CO2 incubator for 24h and 48h respectively; take out the 96-well plate after 24h and 48h, discard the original culture medium, add 100μL basal medium to each well, and then add 10μL CCK -8 indicator, shake gently to mix, place in a 37°C, 5% CO ₂ incubator and incubate in the dark for 2 hours; after 2 hours, take out the 96-well plate, use a microplate reader to detect the OD value of each well at a wavelength of 450 nm, and calculate each Relative proliferation rate of cells in the group.

Experimental results: In this experiment, different concentrations of aptamers T2, Scr and AZD9291 were used on A431 cells and H1975 cells. After 24h and 48h respectively, CCK-8 reagent was added to detect and the relative cell survival rate was calculated. The results are shown in Figure 5. Compared with the negative control Scr group, under the treatment of aptamer T2 at 100 and 200nM concentrations, the relative survival rate of H1975 cells decreased significantly, and the longer the treatment time, the lower the cell survival rate. . However, aptamer T2 showed no inhibitory effect on A431 cells after 24h and 48h. At the same time, the positive control AZD9291 group had a significant inhibitory effect on cell proliferation on H1975 cells and A431 cells. Therefore, it shows that aptamer T2 can specifically affect the survival rate of H1975 cells in vitro and inhibit the proliferation of lung cancer H1975 cells.

Experiment 4: Using the EdU method to explore the effect of aptamer T2 on the proliferation of H1975 cells

When H1975 cells are in the logarithmic growth phase, discard the original culture medium, add PBS to wash once and then add 0.25% trypsin for digestion. When the cells fall off the culture wall, add complete culture medium to terminate digestion. Collect the cells and centrifuge at 15 mL. tube, centrifuge at 900 rpm/min for 5 minutes at room temperature, resuspend the cells in complete culture medium and evenly seed them into a 96-well plate at a density of 5,000 cells per well, and place them in a 37°C, 5% CO2 incubator for culture; The cells in the 96-well plate were randomly divided into three groups, including blank group, negative control group (100nM Scr), positive control group (100nM AZD9291) and experimental group (100nM T2); after discarding the original culture medium in the 96-well plate the next day, use PBS After washing once, add 1640 medium. The experimental group added corresponding concentrations of aptamers T2, Scr and AZD9291, and the blank group only added 1640 medium, and continued to culture for 24 hours in a 37°C, 5% CO2 cell incubator; after 24 hours, the original medium was discarded, and After washing once with PBS, add 100 μl of 50 μM EdU solution to each well, and place it in a 37°C, 5% CO2 cell incubator for 2 hours; discard the medium after 2 hours, and wash three times with PBS for 5 minutes each time; remove the PBS. , add 100 μL of 4% paraformaldehyde solution to each well to fix the cells; after fixing at room temperature for 30 minutes, discard the 4% paraformaldehyde solution in the 96-well plate, add 100 μL of 2 mg/mL glycine to each well, and incubate at room temperature for 5 minutes to neutralize; After 5 minutes of action, discard the glycine solution, add PBS solution and wash twice, add PBS solution containing 0.5% Triton to each well and incubate at room temperature for 10 minutes; discard the original liquid after 10 minutes, wash once with PBS, and add the prepared Apollo staining solution ( Ready to use (recipe provided by RIOBIO kit), incubate at room temperature in the dark for 30 minutes; discard the Apollo staining solution, wash twice with PBS containing 0.5% Triton, 10 minutes each time; use ddH2O to prepare Hoechst 33342 staining solution to detect cell nuclei. Stain, protect from light, and incubate at 37°C for 20 minutes; discard the Hoechst dye solution and wash three times with PBS, 5 minutes each time; after staining, take pictures with a Nikon confocal microscope, and randomly select 15 fields of view from each group for counting and analysis.

Experimental results: After adding T2, Scr, and AZD9291 to a 96-well plate covered with H1975 cells, the cells were treated with drugs for 24 hours, then fixed and stained with EdU for 2 hours, and observed and photographed with a Nikon confocal microscope. The results are shown in Figure 6. Compared with the blank group (Control) and the negative control group (Scr), the fluorescence intensity of H1975 cells after aptamer T2 and AZD9291 were administered for 24 hours was significantly reduced. Through relative proliferation rate analysis, it can be seen that the relative proliferation rates of the aptamer T2 group and the positive control group were significantly reduced, which had a similar effect of inhibiting H1975 cell proliferation. The above results indicate that aptamer T2 can inhibit the proliferation of lung cancer H1975 cells.

Experiment 5. Scratch experiment to detect the effect of aptamer T2 on the migration of lung cancer H1975 cells

Cell preparation: When H1975 cells and A431 cells are in the logarithmic growth phase, discard the original culture medium, add PBS to wash once and then add 0.25% trypsin for digestion. When the cells fall off the culture wall, add complete culture medium to terminate digestion. , collect the cells into a 15mL centrifuge tube, centrifuge at 900 rpm/min for 5 min at room temperature, resuspend the cells in complete culture medium, and seed them evenly into a six-well plate at a density of 1×10 ⁵ cells per well, and place at 37°C in 5% CO ₂ Culture in an incubator; cell starvation treatment: When the cell density reaches 80%-90%, remove the original medium, wash once with PBS, add cell culture medium containing 2% FBS, and starve for 6 hours to remove the effect of serum on cell proliferation. Effect; Scratch: After cell starvation treatment, use a 200 μL pipette tip perpendicular to the six-well plate to scratch the cells, and each well should be scored straight into a cross. Discard the original culture medium and slowly rinse it once with basal medium to wash off the fallen cells; randomly divide the scratched cells into 4 groups: blank group (no treatment, add 1640 and DMEM basal medium respectively) , experimental group (200nM concentration of aptamer T2 treated for 24h), negative control group (200nM concentration of aptamer Scr treated for 24h), positive control group (50nM concentration of AZD9291 treated for 24h); discard the original culture medium, and add The corresponding concentrations of aptamers T2, Scr and AZD9291 were prepared in the basal medium. The blank group was added with 1640 and DMEM basal medium respectively. Each group was set up with 3 parallel wells. After drug administration, culture in a 37°C, 5% _CO2 cell culture incubator; at 0h, 8h and 24h of drug treatment, take out the six-well plate, observe the scratches under a microscope and take photos, and calculate the relative scratch healing of each group. Rate.

Experimental results: By calculating the scratch healing area, the relative migration ability of cells under drug administration can be judged. The results are shown in Figure 7A. Compared with the blank group (Control) and the negative control group (Scr), the experimental group (T2) and the positive control group (AZD9291) acted on lung cancer H1975 cells after scratching, and their relative healing areas were significantly reduced. It was calculated that the cell migration rate at 8h and 24h showed a significant downward trend compared with the Control group and Scr group, and the difference was statistically significant (Figure 7B). Figure 7C shows that each group's administration acts on A431 cells after scratching. The relative healing area of each group has little change. After statistical analysis, the aptamer T2 group has no significant difference compared with the blank group and the negative control group (P>0.05 ), while the relative scratch healing area rate of the positive control group (AZD9291) on A431 cells after scratching was statistically different (P<0.05) (Figure 7D). The results show that aptamer T2 can specifically inhibit the migration ability of lung cancer H1975 cells.

Experiment 6. Transwell experiment to detect the effect of aptamer T2 on the migration ability of H1975 cells

When H1975 cells are in the logarithmic growth phase, discard the original culture medium, wash once with PBS, then add cell culture medium containing 2% FBS and starve for 6 hours; take the Transwell chamber and place it in a 24-well plate. Add 500 μL of 1640 medium to the upper chamber and place it in a 37°C incubator for 2 hours to hydrate the basement membrane; after starving the cells for 6 hours, remove the original medium, wash once with PBS, digest the cells with 0.25% trypsin, and add complete medium After digestion is terminated, collect the cells into a 15mL centrifuge tube and centrifuge at 900rpm/min for 5 minutes at room temperature. Remove the supernatant and resuspend the cells in 1640 culture medium for later use. Control the cell density to 1×10 ⁵ /mL; take out the Transwell chamber and discard the original culture medium. , add 200μL cell suspension to each Transwell chamber. The Transwell chambers seeded with cells were randomly divided into three groups: blank group, experimental group (treated with aptamer T2 at a concentration of 200 nM for 24 hours), and control group (treated with aptamer Scr at a concentration of 200 nM for 24 hours). Add corresponding concentrations of aptamers T2 and Scr in the Transwell chamber, and the blank group will not receive any treatment; add 600 μL of 1640 culture medium containing 20% FBS to the lower chamber of the Transwell, and gently tap the edge of the 24-well plate to eliminate air bubbles. Influence. Place it in a 37°C, 5% _CO2 cell culture incubator for 24 hours; after adding drugs and culturing for 24 hours, take out the 24-well plate, remove the original culture medium in the Transwell chamber and the lower chamber, add 600 μL of 4% paraformaldehyde in the lower chamber, and keep at room temperature. Fix the cells for 30 minutes; remove the 4% paraformaldehyde solution after the cells are fixed, add PBS to the lower chamber and wash 3 times, 5 minutes each time, gently wipe the chamber with a sterile cotton swab to remove cells adhered to the inner wall of the chamber; add 600 μL to the lower chamber Dye the crystal violet dye solution at room temperature for 30 minutes. Recover the crystal violet dye solution. Wash the chamber with ddH ₂ O for 5 minutes each time. Repeat washing three times and then dry the Transwell chamber at room temperature. Observe and take photos with a microscope, and randomly select 9 cells. Field of view for cell counting.

Experimental results: The Transwell migration experiment consisted of a blank group (Control), a control group (Scr, AZD9291) and an experimental group (T2). After the cells were starved for 6 hours, corresponding concentrations of aptamers T2, Scr and AZD9291 were added to treat lung cancer H1975 cells. After 24 hours, the number of cells in a single field of view of cells that passed through the basement membrane of the Transwell chamber was counted. The results shown in Figure 8 show that after 24 hours of lung cancer H1975 cell administration, there was a significant difference in the migration ability of the cells between the blank group (Control), the experimental group (T2) group, and the positive control (AZD9291) group (P<0.001). , while compared with the control group (Control) and the random control (Scr) group, there was no significant difference in the migration ability of cells. The above results suggest that aptamer T2 can inhibit the migration ability of lung cancer H1975 cells.

Experiment 7. Transwell experiment to detect the effect of aptamer T2 on the invasion ability of H1975 cells

Dilute Matrigel 1:5 with serum-free 1640 culture medium, then spread 50 μL of diluted Matrigel evenly inside the transwell chamber, and let it stand for 30 minutes in a 37°C incubator to polymerize into a gel; remove the Matrigel to solidify. Add 50 μL of serum-free DMEM culture medium to the water precipitated during the gelation process, and place it in a 37°C incubator for 30 minutes to hydrate the basement membrane; other steps are the same as those in Experiment 6 Transwell migration experiment.

Experimental results: The Transwell invasion experiment consisted of a blank group (Control), a control group (Scr, AZD9291) and an experimental group (T2). After the cells were starved for 6 hours, 1640, aptamer T2, Scr and AZD9291 were added respectively. After 24 hours, the cells were examined under the microscope. Observe and count the number of cells that invaded in each group. The experiments in each group were repeated at least three times, and the differences were analyzed. The results shown in Figure 9 show that after 24 hours of lung cancer H1975 cell administration, there was a statistical difference in the number of cells that invaded between the blank group (Control), the experimental group (T2) group, and the positive control (AZD9291) group (P<0.001). , and there is also a significant difference (P<0.001) between the experimental group (T2) group and the positive control (AZD9291) group in the negative control (Scr) group (P<0.001). At the same time, the number of invasions in the blank group (Control) and the negative control group (Scr) The difference is not significant (P>0.05). The above results suggest that aptamer T2 can significantly inhibit the invasion ability of lung cancer H1975 cells.

Experiment 8. Western Blot experiment to explore the molecular mechanism of aptamer T2 on H1975 cells

1. Cell sample preparation: When H1975 cells are in the logarithmic growth phase, discard the original culture medium, add PBS to wash once and then add 0.25% trypsin for digestion. When the cells fall off the culture wall, add complete culture medium to terminate digestion. , collect the cells into a 15mL centrifuge tube, centrifuge at 900 rpm/min for 5 min at room temperature, resuspend the cells in complete culture medium, and seed them evenly into a six-well plate at a density of 1×10 ⁵ cells per well, and place at 37°C in 5% CO ₂ culture in the incubator; when the cell density grows above 80%, discard the original culture medium in the six-well plate, wash once with PBS, add 1640 culture medium containing 2% FBS to starve the cells, and incubate at 37°C, 5% Starve culture for 6 hours in a CO ₂ incubator; cells are randomly divided into three groups: blank group, control group (200nM T2 treatment for 24h) and experimental group (200nM T2 treatment for 24h). Each group has 3 duplicate wells; discard the original cells after 6h. After washing the culture medium once with PBS, the experimental group and the control group respectively added 1 mL of 1640 culture medium to prepare aptamers T2 and Scr with a final concentration of 100 nM. The blank group only added 1 mL of 1640 culture medium and incubated at 37°C and 5% CO ₂ Culture in cell culture incubator for 24h.

2. Extract cell proteins: After 24 hours, take out the six-well plate and place it on ice, discard the complete culture medium, and wash once with 4°C pre-cooled PBS to remove residual culture medium; add 100 μL lysis solution to each well, and use clean cells Scrape off each group of cells with a scraper and collect them into a 1.5 mL EP centrifuge tube; ultrasonic lysis: 25% amplitude, 0.5 s/time, ultrasonic for 10 s in ice bath, let stand for 10 s, repeat 10 times; rotate and shake at 4°C Lyse in bed for 30 minutes, centrifuge at 14000 rcf for 10 minutes at 4°C, collect the supernatant in a new EP tube, and store at -20°C.

3. Determination of protein concentration by BCA method: Protein quantification is performed according to the instructions of the BCA Protein Assay Kit of Thermo Fisher Scientific. Preparation of the standard curve: Dilute the bovine serum albumin standard solution with a concentration of 2 mg/mL in the kit 6 times according to the table. , make a standard curve.

Table 6 BCA standard protein solution dilution table

Prepare working solution: Prepare BCA reaction working solution (A solution: B solution = 50:1) and place it on ice; Color development reaction: Take a 96-well plate, delineate the sampling holes, and set three duplicate holes in each group. Add 100 μL of working solution to each well; add 5 μL of the diluted standard and the protein sample extracted in step 1.5.2 into the working solution in sequence; place the 96-well plate at 37°C and incubate for 30 minutes; take out the 96-well plate and use The microplate reader measures the OD value of the protein sample at a wavelength of 595 nm, and the concentration of the protein sample to be tested is calculated through the standard curve method.

4. SDS-PAGE electrophoresis, gel preparation: Assemble the gel mold, fill the glass plate with ddH ₂ O to check for leakage, prepare 10% separation gel according to the formula; gel filling: pour out the ddH ₂ O in the glass plate, Absorb excess moisture from the outside through filter paper siphoning. Add an appropriate amount of the prepared separation gel solution into the glass plate. The interface is controlled at 2/3 of the glass plate, and absolute ethanol is added to the separating gel solution to flatten the liquid level. Let it stand at room temperature for more than 30 minutes. Observe that the separation gel appears with a clear layered interface, indicating that the gel has solidified. Prepare 5% concentrated gel according to the formula, turn the gel dispensing device upside down, and pour out the absolute ethanol. Slowly add 5% concentrated glue solution and insert the comb. Pay attention to prevent liquid splashing and bubbles during the entire operation. Let stand at room temperature for 30 minutes until the concentration gel solidifies. Remove the glass plate and place it at 4°C for later use. Sample loading: According to the protein sample concentration measured by BCA, use RIPA solution to adjust the protein concentration to ensure that the loading amount of each well protein sample is 20 μg. . Add SDS-PAGE reducing loading buffer (5× loading buffer) and protein sample and mix in a ratio of 1:4, denature in a metal bath at 99°C for 10 minutes, and cool on ice for 5 minutes. Assemble the electrophoresis equipment, prepare the electrophoresis buffer, add the electrophoresis solution into the tank, pull out the comb vertically, and add the marker and protein sample to the loading hole; electrophoresis: connect the power supply, the electrophoresis condition of the stacking gel is 80V, wait until the sample runs to the lower layer When separating the gel, the electrophoresis condition is 120V constant voltage, and the power supply is cut off when the sample reaches the bottom of the gel.

5. Membrane transfer: Prepare the electroporation buffer, place it in a 4°C refrigerator to pre-cool, and prepare the PVDF membrane for soaking in methanol solution for 2 minutes for activation. After electrophoresis stops, remove the PAGE gel from the glass plate; assemble the transfer sandwich: transfer the sandwich to the membrane Soak the clip flatly in the electroporation buffer, with the black side facing down. Place 2 layers of sponge, 2 pieces of filter paper, PAGE gel, PVDF membrane, 2 pieces of filter paper, and 2 layers of sponge pads in order. After the assembly is completed, slowly combine the sandwich transfer clips; electroconversion: insert the assembled sandwich transfer clip vertically into the electrotransfer tank. Note that the black surface of the transfer clip must face the black negative electrode in the tank. Fill the electroporation buffer with 320mA constant current and transfer the membrane for 180 minutes. During the transfer, an ice bath is required to cool down the entire process.

6. Antibody binding reaction: Blocking: Take out the transferred PVDF membrane, apply blocking solution (prepare 5% skim milk powder according to the formula as blocking solution), place it on a shaker and block at room temperature for 2 hours; incubate the primary antibody: pour out the blocking solution after 2 hours solution, add TBST solution to wash the membrane 3 times, 5 minutes each time. Cut the membrane according to the target band, add the target protein and internal reference protein antibodies, and incubate at 4°C overnight; incubate the secondary antibody: recover the antibodies the next day, and wash the membrane 3 times with TBST solution, 5 minutes each time. Horseradish peroxidase-labeled IgG corresponding to the source of the antibody was added. Incubate at room temperature for 1 hour; discard the secondary antibody solution after 1 hour, and wash the membrane 3 times with TBST solution, 5 minutes each time.

7. Development: Prepare a chemiluminescent liquid, apply the chemiluminescent liquid to the PVDF membrane, and develop it with a Bio-Rad gel imager.

Experimental results: In preliminary work, it was verified that aptamer T2 can specifically target and bind to lung cancer H1975 cells expressing EGFR T790M and can effectively inhibit the proliferation and migration of H1975 cells. In order to further explore the mechanism of aptamer T2 inhibiting the proliferation of H1975 cells, a blank group (Control), a control group (Scr) and an experimental group (T2) were established. After H1975 cells were starved for 6 hours, 200nM T2 and Scr were added to act on H1975 cells respectively. , 24 hours later, cellular proteins were extracted and Westernblot experiments were performed to detect the phosphorylation levels and total protein level expression of EGFR and AKT. The results can be seen in Figure 10. Comparing the blank group (Control) and the control group (Scr), after 24 hours of aptamer T2 treatment, the EGFR phosphorylation level of H1975 cells decreased (P<0.01) while the expression level of total protein EGFR remained unchanged. Phosphorylated EGFR continuously activates downstream signal transduction pathways, triggers gene transcription in turn, and causes a series of cascade reactions to control cell proliferation and inhibit apoptosis. The results show that aptamer T2 may function by inhibiting the autophosphorylation of EGFR and AKT, thereby inhibiting cell proliferation.

Experiment 9. To explore the in vivo tumor inhibitory effect of aptamer T2 in nude mice with lung cancer subcutaneous tumor model.

experimental method:

1. Establishment of lung cancer subcutaneous tumor model in nude mice: H1975 cells were cultured in 1640 containing 10% FBS and expanded in a 37°C, 5% CO2 incubator. 3- to 4-week-old male BALA/c were purchased from the Guangdong Provincial Animal Center. Nude mice were raised in an SPF environment for one week; H1975 cells that were growing well and in the logarithmic growth phase were taken, washed once with PBS, digested with 0.25% trypsin, centrifuged at 900 rpm/min for 5 minutes at room temperature, discarded the supernatant, and washed once with PBS Remove the remaining serum from the cells; resuspend the cells in PBS, control the cell density to 1 to 2×10 ⁶ cells/mL, place the cells in an ice box, and store them for later use; use a 1mL sterile syringe to draw the cell suspension , BALA/c nude mice were injected subcutaneously, each nude mouse was injected with 100 μL of cell suspension, and the injection site was the buttocks of the hind limbs. After injection, the tumor growth in nude mice was observed and the tumor volume was measured with a vernier caliper. The tumor volume was V=1/2 (a×b ² ) (a is the long diameter of the tumor, b is the short diameter of the tumor).

2. Intratumoral injection and administration: average grouping: nude mice with successfully constructed lung cancer subcutaneous tumor models were randomly divided into three groups, namely the blank group (NaCl), the control group (Scr) and the experimental group (T2), each group had 6 Only; Intratumoral administration: Intratumoral injections were performed on nude mice with microsyringes. The experimental group and the control group were injected with aptamers T2 and Scr (once every other day, 400pmol each time, 6 times in total), and the blank group was injected with aptamers T2 and Scr respectively. Inject the same volume of saline solution. The body weight and tumor volume of the nude mice were measured, and the survival time of the nude mice was recorded. After the nude mice died, the tumor tissue was surgically removed.

Experimental results: 4-week-old male BALA/c nude mice were subcutaneously injected with lung cancer H1975 cell suspension to establish a lung cancer tumor-bearing mouse model. After the tumors were formed, they were randomly divided into a normal saline group (NaCl), a control group (Scr) and an experimental group (T2). The experimental group and the control group were injected intratumorally with 400pmol T2 and 400pmol Scr every other day. The normal saline group was intratumorally injected. Inject the same volume of saline. After the administration, the survival time of the lung cancer model nude mice was recorded, and after death, the tumor tissue was surgically removed and the tumor volume was measured. The experimental results are shown in Figures 11A and 11B. The tumor volume of nude mice in the experimental group (T2) was reduced compared with the NaCl group and Scr group, and the difference was statistically significant. At the same time, comparing the survival curves of the normal saline group and the aptamer T2 group (11C and D), it can be seen that the survival time of the aptamer T2 group is longer than that of the normal saline group and the control group (P<0.05). The above results indicate that intratumoral injection of aptamer T2 has a tumor suppressive effect in the lung cancer tumor-bearing mouse model and can prolong the survival time of the model nude mice.

Claims (5)

1. A nucleic acid aptamer that specifically binds to EGFR T790M mutant protein, characterized in that the sequence is as follows: T2: 5'-ATCCAGAGTGACGCAGCATTTTGACGCTTTATCCTTTTCTTATGGTGGGATAGTTTCGTGGACACGGTGGCTTAGT-3'. 2. Application of the nucleic acid aptamer according to claim 1 in the preparation of drugs that inhibit the proliferation, migration and invasion of lung cancer H1975 cells expressing EGFR T790M mutant protein. 3. Application of the nucleic acid aptamer according to claim 1 in the preparation of drugs for inhibiting the proliferation of subcutaneous lung cancer tumors. 4. A drug for treating lung cancer, characterized in that it contains the nucleic acid aptamer according to claim 1 as an active ingredient. 5. The drug for treating lung cancer according to claim 4, wherein the nucleic acid aptamer contains pharmaceutically acceptable excipients.