CN107602670A - It is a kind of can antagonism EWSR1 protein rna binding activity polypeptide EIP 22 and its application - Google Patents

Abstract

The invention provides it is a kind of can antagonism EWSR1 protein rna binding activity polypeptide and its application, its amino acid sequence such as SEQ ID NO:Shown in 1；A kind of antineoplastic polypeptide and its application are further related to, the antineoplastic polypeptide includes tumor cytotoxicity domain and wears spanning domain, the amino acid sequence such as SEQ ID NO of tumor cytotoxicity domain:Shown in 1.The spanning domain of wearing of the antineoplastic polypeptide of the present invention does not have cytotoxicity in itself, but after connection tumor cytotoxicity domain, there is the obvious effect for suppressing tumor proliferation, migrating invasion and attack.The antineoplastic polypeptide of the present invention, it can not only be expected to combine other treatment mode to suppress tumour separately as antitumor biotherapeutics.

Description

A polypeptide EIP-22 capable of antagonizing the RNA binding activity of EWSR1 protein and its application

technical field

The invention relates to the field of tumor targeting therapy, and more particularly relates to a polypeptide capable of antagonizing the RNA binding activity of EWSR1 protein and its application.

Background technique

Cancer is a major public health problem in the world and has been widely concerned by people. According to the reports of the International Union Against Cancer (UICC) and the World Health Organization (WHO), the global cancer incidence and mortality have continued to rise in recent years. With the continuous growth of the population and the appearance of aging, there are increasing cases of death from malignant tumors every year.

The incidence of cancer worldwide is gradually increasing. According to statistics, 1 out of every 8 deaths is cancer. The grim reality forces humans to constantly seek new ways to treat cancer. Due to the significant individual differences in tumors, traditional medical methods have great limitations in tumor treatment. At present, new treatment options include immunotherapy, targeted therapy, etc. The latter is to design corresponding therapeutic drugs for the identified carcinogenic sites at the cellular molecular level, so as to selectively kill tumor cells without affecting normal tissue cells. The development of targeted therapy has made the treatment of human cancer more precise. In the past ten years, the research and development of targeted drugs has accounted for 80% of the research and development of tumor drugs, including small molecule inhibitors and monoclonal antibodies.

Due to the advantages of easy synthesis, low immunogenicity, high activity, mild adverse reactions and wide sources, targeting peptides have been applied in the research of antitumor drugs. Anti-tumor polypeptides play a role in inhibiting the occurrence and development of tumors by binding to specific sites. So far, tens of thousands of peptides have been discovered in organisms, which are widely involved in regulating the functional activities of various systems, organs, tissues and cells in the body, and play an important role in life activities. In recent years, peptide drugs synthesized by modern biotechnology have become one of the hot spots in drug research and development.

EWSR1 (EWS RNA binding protein 1) gene encodes a multifunctional protein involved in various cell biological processes, including N-terminal transcription activation domain and C-terminal RNA binding domain. Translocations between the EWSR1 gene and a variety of chromosomes encoding transcription factors can produce chimeric proteins that promote tumor development. It is known that EWSR1 is closely related to various tumors, such as Ewing's sarcoma, chondrosarcoma, acute leukemia, neuroblastoma, etc., and EWSR1 plays a role in promoting the occurrence and development of these tumors. So far, EWSR1-specific inhibitors have not been reported.

Contents of the invention

In order to solve the above problems, we tried to block the tumor-promoting effect of EWSR1 by designing targeting polypeptides to target the action site of EWSR1, and finally inhibit the occurrence and development of tumors. During the research process, the inventors prepared a biologically active peptide, and linked the peptide with a cell-penetrating peptide through a covalent bond to achieve the effect of targeting tumor cells and efficiently entering cells.

Based on this study, the present invention provides a polypeptide capable of antagonizing the RNA binding activity of EWSR1 protein, the amino acid sequence of which is shown in SEQ ID NO:1. Experiments have proved that the polypeptide can competitively antagonize the combination of the tumor-promoting gene EWSR1 and RNA, and exhibit obvious tumor suppressing effect at the cell level.

The present invention also provides the application of the above-mentioned polypeptide capable of antagonizing the RNA binding activity of EWSR1 protein in the preparation of antitumor drugs.

The present invention also provides an anti-tumor polypeptide, which includes a tumor cell killing domain and a membrane-penetrating domain, and the amino acid sequence of the tumor cell killing domain is shown in SEQ ID NO:1.

Preferably, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO:2.

Preferably, the transmembrane domain is connected to the N-terminal of the tumor cell killing domain.

The present invention also provides the application of the above-mentioned anti-tumor polypeptide in the preparation of anti-tumor drugs.

The novel polypeptide designed in the present invention specifically and competitively inhibits the combination of EWS1 and RNA, thus suppressing the biological effects brought about by the interaction between the two, and exerting the effect of inhibiting tumors. In addition, a membrane-penetrating peptide can be added to the N-terminal of the polypeptide to increase its efficiency of entering cells. The addition of biotin labeling makes it more convenient to apply at the cellular level, and the experiment enriches related molecules through biotin, so as to carry out the next step of mechanism research. The addition of fluorescein isothiocyanate (FITC) labeling enables the observation of the location of the polypeptide molecule in the cell through a fluorescent confocal microscope, and clarifies the region where it plays a role.

Description of drawings

Figure 1 is a schematic diagram of the principle of EIP-22 competitively antagonizing the interaction between EWSR1 protein and RNA;

Fig. 2 is the fluorescent micrograph of control peptide and EIP-22 treatment HeLa cell after 48 hours;

Fig. 3 is the MTT colorimetric statistical diagram of treating HeLa cells with different concentrations of EIP-22 or control peptides;

Fig. 4 is the MTT colorimetric statistical diagram of HeLa cells treated with EIP-22 or control peptide for different time;

Fig. 5 is the photo of plate colony formation experiment;

Fig. 6 is a statistical diagram of the number of cell clones calculated according to Fig. 5;

Figure 7 is a photo of Transwell cell invasion experiment;

Fig. 8 is a statistical diagram of the migration number of tumor cells calculated according to Fig. 7;

Figure 9 is a photo of RNA co-immunoprecipitation experiment (RIP);

Figure 10 is a photograph of the Peptide pull down experiment.

detailed description

The principles and features of the present invention are described below in conjunction with examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention.

1. Synthesis of Antitumor Peptides

Synthesize an anti-tumor polypeptide by solid-phase synthesis, which includes a tumor cell killing domain and a membrane-penetrating domain, wherein the tumor cell killing domain sequence is shown in SEQ ID NO: 1, and the membrane-penetrating domain sequence is as shown in SEQ ID NO: 1 As shown in ID NO: 2, it is connected to the N-terminal of the tumor cell killing domain, and the obtained sequence is: the amino acid sequence is YGRKKRRQRRR-RTGQPMIHIYLDKETGKPKGDK (SEQ ID NO: 3), named EIP-22. For the convenience of research, we linked FITC labeled with fluorescein isothiocyanate to the C-terminus of the anti-tumor polypeptide, and linked biotin-labeled Biotin to the N-terminus.

The tumor suppressor polypeptide EIP-22 synthesized by the company has been tested by High Performance Liquid Chromatography (HPLC), and its purity has reached 97%. Dissolve the peptide in sterile PBS buffer at a storage concentration of 1mmol/L, dispense it into 1.5ml light-proof EP tubes with a final concentration of 200μmol/L, and store all in the dark at -80°C for later use.

The principle of EIP-22 competitively antagonizing the interaction between EWS1 protein and RNA is shown in Figure 1.

2. Detection of cellular localization of EIP-22

HeLa cells in the logarithmic growth phase were taken and incubated with medium containing EIP-22. Use laser confocal microscopy to dynamically observe the efficiency of the polypeptide being taken up by cells, and the results are shown in Figure 2: Since the 3'-end of the polypeptide has a FITC group, it is possible to observe the situation of the polypeptide entering the cell, and it is found that after adding the polypeptide for 48 hours , the uptake rate of the polypeptide by cells is 90%. In addition, we use DiI (red dye for cell membrane) to observe the cell membrane, so as to objectively and concisely distinguish the nucleus and cytoplasm, and observe the situation of the polypeptide entering the cell more clearly. We found that the polypeptide EIP-22 is located in In the nucleus, it has the same localization as the EWSR1 molecule. It can be seen that the polypeptide can efficiently enter cells and is located in the same nucleus as EWSR1.

3. MTT colorimetric assay to detect the inhibitory effect of EIP-22 on tumor cell growth

HeLa cells in the logarithmic growth phase were taken and incubated with medium containing EIP-22. Carry out MTT experiment, lgIC50=Xm-I(P-(3-Pm-Pn)/4), wherein, Xm=lg maximum dose, I=lg (maximum dose/adjacent dose), P=positive reaction rate sum , Pm = maximum positive reaction rate, Pn = minimum positive reaction rate. From this calculation, the IC50 of the polypeptide EIP-22 was 25 μmol/L.

Based on the IC50 obtained from the above experiments, MTT experiments were further carried out to detect the effect of the polypeptide on the proliferation ability of tumor cells (HeLa cells used in this experiment) at different times and at different concentrations of the polypeptide.

The results are shown in Figures 3 and 4. Compared with the control polypeptide, the viability of tumor cells treated with EIP-22 polypeptide was significantly reduced, and the effect was time- and dose-dependent, indicating that the new polypeptide can effectively reduce the viability of tumor cells. Vitality; when the cells are treated for the same time, the cell viability inhibition rate is higher when the polypeptide concentration is 20-30 μmol/L; when the cell concentration is the same, the cell viability inhibition rate is higher when the polypeptide treatment time is 48-72 hours.

4. Plate colony formation assay to detect the inhibitory effect of EIP-22 on tumor cell proliferation

Take a monolayer of cells in the logarithmic growth phase and inoculate them in a 6-well plate with a cell density of about 1000 cells/well. After the cells adhere to the wall, add appropriate EIP-22 and control to the cells the next day according to the IC50 obtained in the previous stage. Peptides, continue to culture the cells, and constantly observe the growth of the cells. When the clones visible to the naked eye appear in the culture dish, the culture is terminated. After washing with PBS, fix for 20-30 minutes. Then remove the fixative, add an appropriate amount of Coomassie Brilliant Blue for staining, and stain for about 20-30 minutes, then slowly wash away the staining solution with running water, and air dry.

The experimental results are shown in Figures 5 and 6. Compared with the control polypeptide, the clonal colony formation of tumor cells treated with EIP-22 polypeptide was significantly reduced, and the effect was dose-dependent, indicating that the new polypeptide can effectively inhibit tumor cell growth. proliferative capacity.

5. Transwell assay to detect the effect of EIP-22 on the migration activity of tumor cells

Transfer EIP-22 and control peptides into the cells respectively, digest the cells 24 hours later, add a sterile Transwell chamber to the 24-well plate, add 500 μl of medium containing 10% FBS to the lower layer of the chamber, and add the treated cells to the chamber, And about 200 μl of serum-free medium was added for cell culture, so as to observe the effect of tumor suppressor polypeptide on tumor cells.

The results are shown in Figures 7 and 8, compared with the control polypeptide, the migration number of tumor cells treated with EIP-22 was significantly reduced, indicating that the new polypeptide can effectively inhibit the migration ability of tumor cells.

6. EIP-22 inhibits tumor mechanism

The above experiments show that the tumor suppressor polypeptide EIP-22 designed in the present invention can inhibit the proliferation, migration and biological behavior of tumor cells. We assume that the polypeptide exerts the tumor suppressor effect by competitively antagonizing the combination of RNA and EWSR1. In order to verify this hypothesis, we used RNA co-immunoprecipitation (RIP) technology to detect the effect of the tumor suppressor polypeptide EIP-22 on the binding ability of EWSR1 to RNA. The specific steps are as follows:

The EWSR1-RNA complex was precipitated using the EWSR1 antibody, purified and separated to obtain the corresponding RNA samples, and the primers for qRT-PCR were designed to check the changes in the RNA expression content. The results are shown in Figure 9, EIP-22 can significantly inhibit the binding of EWSR1 to RNA.

In the case of tumor cells overexpressing the target RNA, the tumor suppressor polypeptide EIP-22 and the control peptide were added, and the peptide pull down experiment was carried out by using the biotin label added to the N-terminal of the polypeptide. Collect the cells added with the tumor suppressor polypeptide EIP-22 and the control peptide in the culture dish, the cell volume reaches 10 ⁸ , prepare fresh cell lysates, divide them into three groups of Input, IgG, and RNA, and combine them with RNA probes and magnetic Bead incubation, elution and other steps, the final collected protein samples were subjected to western blot. The results are shown in Figure 10, the tumor suppressor polypeptide can significantly inhibit the combination of RNA and EWSR1, indicating that EIP-22 achieves its anti-tumor effect by competitively antagonizing the combination of EWSR1 and RNA.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

sequence listing

<110> Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology

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Claims (6)

1. it is a kind of can antagonism EWSR1 protein rna binding activity polypeptide, it is characterised in that amino acid sequence such as SEQ ID NO:1 It is shown.

2. described in claim 1 can antagonism EWSR1 protein rna binding activity polypeptide in antineoplastic is prepared should With.

3. a kind of antineoplastic polypeptide, it is characterised in that including tumor cytotoxicity domain and wear spanning domain, the tumour is thin Born of the same parents kill the amino acid sequence such as SEQ ID NO of domain:Shown in 1.

4. antineoplastic polypeptide according to claim 3, it is characterised in that the amino acid sequence for wearing spanning domain is such as SEQ ID NO:Shown in 2.

5. the antineoplastic polypeptide according to claim 3 or 4, it is characterised in that the spanning domain of wearing is connected to described swell The N-terminal of cytotoxic effect domain.

6. application of the antineoplastic polypeptide any one of claim 3-5 in antineoplastic is prepared.