CN107446023A - It is a kind of can antagonism HuR protein rna binding activity polypeptide HIP 13 and its application - Google Patents

Abstract

The present invention provides a polypeptide that can antagonize the RNA binding activity of HuR protein and its application, and its amino acid sequence is shown in SEQ ID NO: 1; it also relates to an anti-tumor polypeptide and its application, and the anti-tumor polypeptide includes tumor cells The killing domain and the membrane-penetrating domain, the amino acid sequence of the tumor cell killing domain is shown in SEQ ID NO:1. The transmembrane domain of the anti-tumor polypeptide of the present invention has no cytotoxicity itself, but after linking with the tumor cell killing domain, it has obvious effects of inhibiting tumor proliferation, migration and invasion. The anti-tumor polypeptide of the present invention can not only be used as an anti-tumor biotherapeutic drug alone, but is also expected to be combined with other treatment methods to inhibit tumors.

Description

A polypeptide HIP-13 capable of antagonizing HuR protein RNA binding activity and its application

technical field

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

Background technique

Cancer is the main cause of human death and a major disease that endangers human health. According to the International Agency for Research on Cancer, there were 14.1 million new cases and 8.2 million deaths worldwide in 2012. Although in the past few decades, due to the advancement of surgery, radiotherapy and chemotherapy, tumor treatment has made great progress, but these methods often have severe side effects during treatment, and most cancers still have a high recurrence rate and death risk. Therefore, it is very necessary and urgent to seek new treatment measures to improve the survival rate and cure rate of cancer.

Molecular targeted therapy, which has emerged in recent years, aims at the molecular level to design corresponding therapeutic drugs for the identified carcinogenic targets, specifically select the carcinogenic targets to play a role, and kill tumor cells without affecting normal tissue cells. In recent years, new molecular targeted drugs have achieved remarkable curative effects in clinical practice, pushing the treatment of cancer to a new stage. Targeting peptides are currently considered to be an ideal molecular targeted therapy for tumors. Searching for short peptides that may specifically bind to some special genes in tumor cells can achieve the purpose of targeted therapy for tumors.

Human antigen R (human antigen R, HuR) is a member of the embryonic lethal abnormal vision (ELAV) protein family of RNA-binding proteins, and is highly expressed in a variety of human tumor tissues and cells, such as breast cancer, gastric cancer, colorectal cancer, cervical cancer Carcinoma, ovarian cancer and lung cancer, associated with tumor progression, metastasis and prognosis. HuR can regulate the mRNA stability of growth factors and cytokines, such as p21, c-fos, c-Myc, p53, cyclinA, cyclin E1, COX-2, and regulate tumor cell growth, proliferation and apoptosis. The active form of HuR is positively correlated with the malignancy of various tumors. Antitumor drugs targeting HuR will provide new ideas for tumor treatment and bring new breakthroughs in the diagnosis and treatment of tumors. However, there are no reports of antitumor drugs targeting HuR.

Therefore, it is necessary to construct a novel polypeptide that can not only target tumor cells, but also enter cells efficiently.

Contents of the invention

In order to solve the above problems, the inventors prepared a bioactive peptide, and linked the peptide with a cell-penetrating peptide through a covalent bond, so as to not only target tumor cells, but also have the effect of efficiently entering cells.

Based on this study, the present invention provides a polypeptide capable of antagonizing the RNA binding activity of HuR 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 cancer-promoting gene HuR 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 HuR 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 advantage of the present invention is that the transmembrane domain of the anti-tumor polypeptide of the present invention has no cytotoxicity itself, but after linking the RNA-binding active peptide of HuR, an obvious tumor-inhibiting effect can be observed at the cell level. The anti-tumor polypeptide of the present invention can not only be used as an anti-tumor biotherapeutic drug alone, but is also expected to be combined with other treatment methods to inhibit tumors.

Description of drawings

Figure 1 is a schematic diagram of the principle of HIP-13 competitively antagonizing the interaction between HuR protein and RNA;

Fig. 2 is the fluorescent micrograph of control peptide and HIP-13 treatment HeLa cell after 48 hours;

Fig. 3 is the MTT colorimetric statistical diagram of processing HeLa with different concentrations of HIP-13 or control peptide;

Fig. 4 is the MTT colorimetric statistical diagram of HeLa cells treated with 20 μmol/L of HIP-13 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);

Fig. 10 is the statistical diagram of PCDHB2mRNA level calculated according to Fig. 9;

FIG. 11 is a statistical chart of SLC2A4 mRNA levels calculated according to FIG. 9 .

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-VAGHSLGYGFVNK (SEQ ID NO: 3), named HIP-13. 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. It was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. and identified by high performance liquid chromatography and mass spectrometry. The purity It is more than 95%. Dissolve the polypeptide in sterile PBS buffer at a concentration of 1 mmol/L, and store it in the dark at -80°C for later use.

The principle of HIP-13 competitively antagonizing the interaction between HuR protein and RNA is shown in Figure 1.

2. Detection of cellular localization of HIP-13

After the HeLa cells were digested with trypsin, the cells were resuspended in the complete medium, blown and blown sufficiently to form a single cell suspension, and counted. The cells were seeded into 24-well culture plates at a density of 20,000 cells/well. When dripping the cell suspension, according to the size of the slide, first drop a small amount of medium in each hole where the slide is to be placed, then place the slide on the drop and press it tightly so that the slide is close to the culture dish. The tension of the media binds them together. After the cells were attached to slides, HeLa cells were incubated with FITC-labeled targeting peptide and control peptide (final concentration: 20 μmol/L), and fixed with 4% paraformaldehyde (prepared in PBS) for 20 minutes after 48 hours. Paraformaldehyde was removed and cells were lightly washed 3 x 3 minutes with PBS. DiI (cell membrane red fluorescent probe, Biyuntian Company, Cat. No. C1036) was used to stain the cell membrane at a final concentration of 5 μl, and the cell nucleus was stained with DAPI dye. position.

The experimental results are shown in Figure 2. HIP-13 can be effectively taken up by cells, and the polypeptide accumulates in the nucleus of tumor cells after 48 hours, showing strong nuclear staining.

3. MTT colorimetric assay to detect the inhibitory effect of HIP-13 on tumor cell growth

HeLa cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 3000 cells/well, and the cells were treated with different concentrations of polypeptides according to the experimental requirements, with 6 replicate wells in each group; the untreated group was set as the control group. Cultivate for different times as needed. Add 20 μl/well of 0.5% MTT to the cancer cells of each of the above groups, continue culturing for 4 hours, discard the supernatant, add 100 μl/well of DMSO, shake at room temperature until the crystals dissolve, and immediately measure the absorbance at a wavelength of 490 nm on a microplate reader ( A) value. Each experiment was repeated 3 times. Cancer cell proliferation activity inhibition rate (%)=(1-average absorbance A value of the experimental group/average absorbance A value of the control group)×100%.

The results are shown in Figures 3 and 4. Compared with the control polypeptide, the viability of tumor cells treated with HIP-13 polypeptide was significantly reduced, and the effect was time- and dose-dependent, indicating that the new polypeptide can effectively reduce the cell 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 15-25 μmol/L; when the cell concentration is the same, the cell viability inhibition rate is higher when the polypeptide treatment time is 24-48 hours

4. Plate colony formation assay to detect the inhibitory effect of HIP-13 on tumor cell proliferation

The monolayer cultured HeLa cells in the logarithmic growth phase were digested with 0.25% trypsin and blown into single cells, and the cells were suspended in the culture medium for later use. After counting the cell suspension, make gradient multiple dilutions, and inoculate in a culture plate at an appropriate cell density of 300 cells/well (it is better to see 5-6 cells per field of view). After the cells were evenly adhered to the wall, the cells were treated according to the needs of the experiment, and an untreated control group was set. Place them in an environment of 37° C., 5% CO ₂ and saturated humidity, and culture them statically for 2-3 weeks. When colonies visible to the naked eye appeared in the culture dish, the culture was terminated, the supernatant was discarded, and carefully soaked twice with PBS. Add pure methanol and fix for 15 minutes. Remove the fixative, add an appropriate amount of Coomassie Brilliant Blue staining solution and stain for 10-30 minutes, wash off the staining solution slowly with running water, and air dry. Take the plate upside down and take a video, count the clones directly with naked eyes or counting software, or count the clones larger than 10 cells under a microscope (low magnification lens).

The experimental results are shown in Figures 5 and 6. Compared with the polypeptide in the control group, the clonal colony formation of tumor cells in the experimental group was significantly reduced after HIP-13 treatment, and the effect was dose-dependent.

5. Transwell assay to detect the effect of HIP-13 on the migration activity of tumor cells

Add 500 μl of medium containing 20% serum to the lower layer of the 24-well culture plate, gently put it on the small chamber, do not generate bubbles, and mix well after counting the cells. The amount added to each well was equal to ensure the same pressure, the cells were treated according to the needs of the experiment, and an untreated control group was set. Incubate overnight at 37°C, 5% CO ₂ and saturated humidity, gently wipe off the upper layer of cells with a cotton swab, add 500 μl of methanol to fix for 10-15 minutes, wash with PBS, stain with crystal violet for more than 15 minutes, wash with PBS, and take pictures , to count the number of cells migrated in each random high power field.

The results are shown in Figures 7 and 8. Compared with the polypeptide in the control group, the migration number of tumor cells in the experimental group was significantly reduced after HIP-13 treatment.

7. The mechanism of HIP-13 inhibiting tumor

RNA co-immunoprecipitation assay (RIP) was used to study the mechanism of HIP-13 inhibiting tumor.

Collect cell lysate: culture HeLa cells in a petri dish, and when the cells grow to 70-80% confluent, add control peptide and HIP-13 to the two groups of cultured cells respectively, after culturing for 48 hours, wash the petri dish twice with cold PBS , after adding cold PBS, scrape the cells with a cell scraper, collect them into EP tubes, centrifuge at 1500rpm, 4°C for 5 minutes, discard the supernatant, collect the cells, resuspend the cells with the same volume of RIP lysate as the cells, pipette evenly and place in Let stand on ice for 5 minutes, dispense 200 μl of cell lysate into each tube, and store at -80°C.

The magnetic beads were resuspended, and the samples included the control peptide and the target sample (Anti-HuR) of the HIP-13 treatment group, negative control (IgG) and positive control (Input). Pipette 50 μl of the resuspended magnetic bead suspension into each EP tube, add 500 μl RIP WashBuffer to each tube, vortex, place the EP tube on the magnetic stand, and turn it left and right 15° to make the magnetic beads adsorb into a straight line, and remove For the supernatant, repeat once, resuspend the magnetic beads with 100 μl of RIP Wash Buffer, and add about 5 μg of the corresponding antibody to each sample. Incubate at room temperature for 30 minutes, place the EP tube on a magnetic stand, discard the supernatant, add 500 μl RIP Wash Buffer, discard the supernatant after vortexing, repeat once, add 500 μl RIP Wash Buffer, vortex and place on ice.

RNA-binding protein immunoprecipitation: Add 900 μl RIP immunoprecipitation buffer to each tube, quickly thaw the cell lysate prepared in the first step, and centrifuge at 14,000 rpm at 4°C for 10 minutes. Pipette 100 μl of the supernatant into the magnetic bead-antibody complex in the previous step to make the total volume 1ml, incubate at 4°C for 3 hours to overnight, centrifuge briefly, put the EP tube on the magnetic stand, discard the supernatant, and add 500 μl of RIP Wash Buffer, vortex and place the EP tube on the magnetic stand, discard the supernatant, and repeat the washing 6 times.

RNA purification and detection: Resuspend the above-mentioned magnetic bead-antibody complex with 150 μl proteinase K buffer, incubate at 55°C for 30 minutes, after incubation, place the EP tube on the magnetic stand, and pipette the supernatant into a new EP tube Add 250μl RIP Wash Buffer to each tube of supernatant, add 400μl phenol: chloroform: isoamyl alcohol to each tube, vortex for 15 seconds, centrifuge at 14000rpm at room temperature for 10 minutes, carefully absorb 350μl of the upper aqueous phase, and inhale To another new EP tube, add 400 μl of chloroform to each tube, vortex for 15 seconds, centrifuge at 14000 rpm for 10 minutes at room temperature, carefully absorb 300 μl of the upper aqueous phase, inhale into another new EP tube, add 50 μl of Salt Solution I to each tube , 15μl Salt Solution Ⅱ, 5μl PrecipitateEnhancer, 850μl absolute ethanol (RNase-free), keep at -80°C for 1h to overnight, centrifuge at 14000rpm, 4°C for 30 minutes, remove the supernatant carefully, wash once with 80% ethanol, 14000rpm, 4°C Centrifuge for 15 minutes, carefully remove the supernatant, dry in the air, dissolve in 10-20 μl DEPC water, store at -80°C, or perform PCR detection after reverse transcription.

The results are shown in Figures 9-11. Compared with the polypeptide of the control group, the HIP-13 polypeptide can significantly reduce the binding level of HuR to RNA, indicating that the polypeptide designed in the embodiment of the present invention can significantly antagonize the ability of HuR to bind to RNA, thereby reducing HuR The function of binding and affecting the biological activity of RNA in cells exerts the function of tumor suppression.

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

<120> A polypeptide HIP-13 capable of antagonizing the RNA binding activity of HuR protein and its application

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<170> PatentIn version 3.5

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Val Ala Gly His Ser Leu Gly Tyr Gly Phe Val Asn Lys

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Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

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Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Val Ala Gly His Ser

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Leu Gly Tyr Gly Phe Val Asn Lys

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Claims (6)

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

2. described in claim 1 can antagonism HuR protein rna binding activity application of the polypeptide in antineoplastic is prepared.

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.