CN107417772A - It is a kind of can antagonism hnRNPU protein rna binding activity polypeptide HIP 20 and its application - Google Patents

Abstract

The present invention provides a polypeptide that can antagonize the RNA binding activity of hnRNPU 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 kind of polypeptide HIP-20 that can antagonize the RNA binding activity of hnRNPU 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 hnRNPU protein and its application.

Background technique

As we all know, tumor is a worldwide medical problem, which brings great harm to human health. Conventional tumor therapy drugs are often poor in tissue specificity, causing damage to normal tissues while killing tumors, causing great side effects and bringing great pain to tumor patients. Therefore, the development of tumor-targeting peptide drugs with high specificity and significant curative effect has aroused great interest.

Tumor-targeting polypeptide refers to a class of active polypeptides that can target specific molecules related to tumor occurrence and development. It is currently considered the most ideal tumor-targeting therapy. Compared with traditional chemotherapy drugs, tumor-targeting peptides have the following advantages: 1. Good tissue penetration, easy to enter tumor cells to play a killing role; 2. High biological activity, strong specificity, low immunogenicity and toxicity Relatively weak; 3. Fast plasma clearance, not easy to accumulate in the body; 4. Less interaction with other drugs, high affinity with in vivo targets; 5. Easy to chemical synthesis. Therefore, in recent years, many scholars have worked hard to find short peptides targeting tumor-specific molecules, so as to achieve the purpose of targeting tumors.

Nuclear heterogeneous ribonucleoproteins (hnRNPs) are a group of RNA-binding proteins involved in various pathophysiological processes such as tumorigenesis, viral infection, and cell apoptosis. Among them, hnRNPU is the phosphorylated protein with the largest molecular weight, which plays an important role in the transcription, localization and expression of genes, especially the apparent inactivation of sex chromosomes. hnRNPU is mostly involved in the regulation of cell functions in the form of DNA/RNA protein complexes, and its scaffold binding region can enhance the function of transcription factors and regulate gene transcription. More and more studies have confirmed that hnRNPU protein is highly expressed in a variety of tumors, and participates in the invasion and metastasis of liver cancer and lung cancer by combining with non-coding RNA in vivo. Although there have been many reports of new anti-tumor polypeptides, some of which have entered clinical trials, there is no report on anti-tumor polypeptides targeting hnRNPU protein.

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 deeply studied the mechanism of action of hnRNPU protein, and found that hnRNPU interacts with long-chain non-coding RNA (LOC101927219) through its RGG domain to promote the transcription of downstream oncogenes or suppress tumor suppressor genes. Blocking the interaction between the RGG domain of hnRNPU protein and lncRNA can effectively inhibit tumor progression.

Based on this research, the present invention provides a polypeptide capable of antagonizing the RNA binding activity of hnRNPU 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 oncogene hnRNPU and LncRNA, and exhibit obvious tumor suppression at the cellular level.

The present invention also provides the application of the above-mentioned polypeptide capable of antagonizing the RNA binding activity of hnRNPU 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 hnRNPU, an obvious tumor-suppressing 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

Fig. 1 is the fluorescent micrographs of HeLa cells and SH-SY5Y cells treated with control peptide and HIP-20 for 48 hours;

Fig. 2 is the MTT colorimetric chart of cells treated with different concentrations of HIP-20 or control peptide;

Fig. 3 is the MTT colorimetric statistical diagram of the cells treated with 20 μmol/L HIP-20 or control peptide at different times;

Fig. 4 is the photo of soft agar colony formation experiment;

Fig. 5 is a statistical diagram of the number of cell clones calculated according to Fig. 4;

Figure 6 is a photo of Transwell cell invasion experiment;

Fig. 7 is a statistical diagram of the migration number of tumor cells calculated according to Fig. 6;

Figure 8 is a photograph of an angiogenesis activity inhibition experiment of tumor cells;

Figure 9 is a statistical diagram of the relative angiogenesis activity of tumor cells;

Figure 10 is a photo of the Peptide pull down experiment;

Figure 11 is a photo of RNA co-immunoprecipitation experiment (RIP).

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-NMRGGNFRGGAPGNRGGYNK (SEQ ID NO: 3), named HIP-20. For the convenience of research, we linked FITC to the C-terminus of the anti-tumor polypeptide.

The synthesis is carried out from the C-terminal to the N-terminal, and the steps are as follows:

a. Weigh n equivalents of resin into the reactor, add DCM (dichloromethane) to swell for half an hour, then remove the DCM, add 2n equivalents of the first amino acid in the sequence, add 2n equivalents of DIEA (diisopropylethylamine ), an appropriate amount of DMF (dimethylformamide), DCM (the appropriate amount refers to that it is advisable to fully agitate the resin), nitrogen bubbling for 60 minutes. Then add about 5n equivalent methanol, react for half an hour, remove the reaction solution, and wash with DMF and MEOH;

b. Add the second amino acid in the sequence (also 2n equivalents), 2n equivalents of HBTU (1-hydroxy, benzo, trichlorazole tetramethylhexafluorophosphate) and DIEA, nitrogen bubbling reaction half to the reactor hours, the liquid was washed off, ninhydrin was detected, and then capped with pyridine and acetic anhydride. Finally, wash, add an appropriate amount of decapping solution to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash, and detect ninhydrin;

c. Adding different amino acids in the sequence in sequence according to step b and performing various modifications;

d. Remove the resin from the reaction column after being blown dry with nitrogen, pour it into a flask, and then add a certain amount of cutting fluid (the ratio of cutting fluid and resin is about 10ml/gram) to the flask (the composition is 95% TFA , 2% ethanedithiol, 2% triisopropylsilane, 1% water), shake, and filter off the resin;

Obtain the filtrate, then add a large amount of diethyl ether to the filtrate to separate out the crude product, then centrifuge and wash to obtain the crude product of the sequence;

Peptide purification: The crude product is purified by reverse-phase HPLC. The peptide is connected to the column filler through hydrophobic interaction, and the ionic strength is gradually reduced for elution. The UV absorbance of the peptide was measured by UV spectrophotometry for quantification.

2. Membrane penetration and cell localization detection of HIP-20

Human neuroblastoma cell line SH-SY5Y and human cervical cancer cell line HeLa were used for experiments. Cells in logarithmic growth phase were planted on coverslips in 24-well plates, with about 8000-10000 cells per well. Culture in 10% fetal bovine serum, high glucose DMEM medium, and add 20 μmol/L polypeptide, maintain for 48 hours. Discard the supernatant, add 4% paraformaldehyde 300 μl/well, fix the cells at room temperature for 20 minutes, discard the paraformaldehyde, wash twice with 1×PBS, 5 minutes each time. Add 300 μl/well of DAPI solution, stain cell nuclei at room temperature for 5 minutes, wash thoroughly with 1×PBS 5 times, 5 minutes each time. Then stain the cell membrane with a cell membrane dye at a concentration of 2 μmol/L and 200 μl/well at room temperature for 5-10 minutes, keep shaking during the staining process, and then fully wash 5 times with 1×PBS, 5 minutes each time. Mount the slides in pure glycerol, and fix the coverslips on slides. Laser confocal microscopy was used to detect the localization of the polypeptide with fluorescent groups in the cells, and the pictures were taken and saved.

The results are shown in Figure 1, after 48 hours after being added to the cell culture system, the polypeptide can effectively enter the tumor cells and show strong nuclear localization. However, the control peptide can only partially enter the cytoplasm, and a very small amount is distributed in the nucleus. It can be seen that the polypeptide can effectively enter tumor cells and localize in the nucleus.

3. MTT colorimetric assay to detect the inhibitory effect of HIP-20 on tumor cell growth

Human neuroblastoma cell line SH-SY5Y was used for colorimetric assay of MTT cell viability. In a 96-well cell culture plate, 5000 cells were planted per well, and 8 replicate wells were set for each sample. After 12 hours, HIP-20 and control peptides were added. Firstly, 0.05, 0.5, 5, 50 μmol/L polypeptide was administered to detect IC50. Secondly, according to the IC50 corresponding to the drug concentration, according to the IC50, the concentration of the polypeptide and its control peptide were 5, 10, 20, and 40 μmol/L; finally, the specific concentration of 20 μmol/L of the polypeptide and the control peptide were given for 12, 24, 48, and 48 μmol/L respectively. 72 hours.

After reaching the calibration time, the supernatant was discarded, and the sample well was washed 3 times with 1×PBS, 5 minutes each time. Then add 100 μl of dimethyl sulfoxide (DMSO) solution to the sample well. The culture plate was left to stand at 37°C for 10 minutes. Gently mix the sample in the well after taking it out. The absorbance at 570 nm was detected with a microplate reader. Collect data, calculate and count.

The results are shown in Figures 2 and 3, compared with the control polypeptide, the viability of tumor cells treated with HIP-20 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 ; Its effect is more obvious when the concentration is 20μmol/L for 48 hours. However, the control peptide did not show obvious growth-inhibiting effect.

4. Soft agar colony formation assay to detect the inhibitory effect of HIP-20 on tumor cell proliferation

The SH-SY5Y and HeLa cells in the logarithmic growth phase were digested with 0.25% trypsin and blown gently to make them into single cells, and the living cells were counted. The number of cells in each well of the six-well plate was controlled at 1000. Two concentrations of low melting point agarose solutions of 1.2% and 0.7% were prepared respectively with distilled water, and after autoclaving, they were maintained at 40° C. without solidification. Add the polypeptide to the medium at a concentration of 20 μmol/L, then mix 1.2% agarose and 2×1640 medium (containing 2×antibiotics and 20% calf serum) at a ratio of 1:1, and take 3ml of the mixture Pour it into a six-hole plate, cool and solidify, and use it as bottom agar and put it in a _CO2 incubator for later use. Mix 0.7% agarose and 2×DMEM medium in a sterile test tube at a ratio of 1:1 to prepare 2ml of supernatant gel, then add 0.2ml of cell suspension to the tube, mix well, and pour into the bottom plate , forming a double agar layer. After the upper layer of agar is solidified, place it in a 37° C., 5% CO ₂ incubator and cultivate it for 21-28 days until it forms cell colonies. Add 0.5% MTT solution to the cultured six-well plate, stain at 4°C for 2-3 hours, take it out for observation and take pictures. Count the number of cell colonies formed.

The results are shown in Figures 4 and 5. Compared with the control polypeptide, the clonal colony formation of tumor cells treated with HIP-20 polypeptide at a concentration of 20 μmol/L was significantly reduced, indicating that the new polypeptide can effectively inhibit the proliferation of tumor cells.

5. Transwell assay to detect the effect of HIP-20 on the migration activity of tumor cells

Place Corning's Transwell chamber (product number: 3422) in a 24-well culture plate, and add complete medium containing 15% fetal bovine serum to the bottom layer. And the control peptide and HIP-20 were added to the medium at a concentration of 20 μmol/L. Prepare a single cell suspension with serum-free medium, after counting, add 200 μl of cell suspension to the upper chamber of the Transwell at a concentration of 8000-20000 cells in 200 μl of serum-free medium. And add the same concentration of polypeptide to the upper chamber as the lower chamber. 5% CO ₂ , 37°C for 24 hours. The small chamber was taken out and fixed in 4% paraformaldehyde for 20 minutes, then put into 0.5% crystal violet staining solution, and stained at room temperature for 2 hours. Gently remove the cells in the upper chamber that have not passed through the porous membrane with a cotton swab, and perform a photographic count of the porous membrane under an inverted microscope.

The results are shown in Figures 6 and 7, compared with the control polypeptide, the migration number of tumor cells treated with HIP-20 was significantly reduced, indicating that the new polypeptide can effectively inhibit the migration ability of tumor cells.

6. Detection of inhibition of tumor cell angiogenesis by HIP-20

The angiogenesis activity of tumor cells can be detected by detecting the growth and angiogenesis status of SH-SY5Y and HeLa cells in extracellular matrix gel; the specific implementation method is as follows, SH-SY5Y and HeLa cells are plated on In a 12-well plate, add 1ml of culture medium to each well, then add 20μmol/L peptide and control peptide; culture at 37°C, 5% CO ₂ for 48 hours, collect the supernatant in the culture well, mark it and freeze it - Set aside at 80°C; take out Matrigel from -20°C, and rewarm in an ice-water bath until it melts. Take a 48-well cell culture plate, add 100 μl Matrigel to the bottom of the well, place it horizontally in a 37°C incubator for 30 minutes, and wait for the Matrigel to solidify; prepare HUVEC single-cell suspension, count, and add 8000 microliters to each well of the 48-well plate -10,000 cells, then add 100 μl of culture supernatant of SH-SY5Y and HeLa cells collected from the control polypeptide and HIP-20 treatment groups; culture at 37°C, 5% CO ₂ for 6-8 hours, and observe dynamically in real time under an inverted microscope The state of tubule formation in HUVEC, photographed in due course.

The results are shown in Figures 8 and 9, compared with the control peptide, the polypeptide at a concentration of 20 μmol/L can significantly inhibit tumor cell angiogenesis.

7. The mechanism of HIP-20 inhibiting tumor

RNA pull-down and RIP assays were used to explore the mechanism of HIP-20 in inhibiting tumors.

HeLa was used to detect the inhibitory effect of peptides on the interaction between LncRNA and hnRNPU protein. Add different concentrations of polypeptides (0, 5, 10, 20 μmol/L) to the cells cultured in a 10 cm culture dish, and set up a 20 μmol/L control polypeptide group at the same time, treat for 48 hours; digest with 0.25% trypsin, collect cells, 1× Wash with PBS and remove the supernatant; use 1ml of RIPA lysate to fully lyse the cell mass, divide the lysate into two equal parts, take 50μl as the Input group and freeze at -80°C, and the remaining 450μl as the RIP group, add 30μl for pre-mixing Magnetic beads and 1 μg LncRNA probe, the other group was added with hnRNPU protein antibody and agarose beads, placed in a 4°C refrigerator and spun overnight at 10 rpm; take out the overnight mixed test tube, centrifuge at 3000 rpm for 1 minute, discard the supernatant, and add 1ml1 Resuspend the beads in ×PBS, centrifuge at 3000rpm for 1 minute, carefully aspirate the supernatant, repeat 4-5 times, wash and discard the part that is not bound to the beads. After the last wash, carefully remove the supernatant, add 40 μl PBS to resuspend the beads; add 10 μl 5×SDS PAGE loading buffer, mix well and bathe in water at 95°C for 10 minutes to dissociate the protein and RNA on the beads; Specific primers for PCR amplification.

Prepare a 12% SDS-PAGE gel, cut out the band of hnRNPU protein (120KDa) according to the molecular weight after electrophoresis, transfer to a membrane to block, and expose after incubation with the corresponding antibody. The inhibitory effect of different concentrations of polypeptides on the interaction between LncRNA and hnRNPU protein was judged according to the light and shade of the band. Prepare 1.5% agarose gel, remove the same volume of PCR products for electrophoresis identification, and conduct semi-quantitative comparison according to the brightness of the electrophoresis bands.

The results are shown in Figures 10 and 11. Using western blot detection, the Input group showed that the protein loading amount of each group was equal, and the RNA pull-down group showed that with the increase of the HOP-20 concentration, the hnRNPU protein bound by the LncRNA probe gradually decreased. , indicating that HIP-20 polypeptide can inhibit the interaction between LncRNA and hnRNPU protein.

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-20 capable of antagonizing the RNA binding activity of hnRNPU protein and its application

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<170> PatentIn version 3.5

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Asn Met Arg Gly Gly Asn Phe Arg Gly Gly Ala Pro Gly Asn Arg Gly

1 5 10 15

Gly Tyr Asn Lys

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<211> 11

<212> PRT

<213> Artificial sequence

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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 Asn Met Arg Gly Gly

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Asn Phe Arg Gly Gly Ala Pro Gly Asn Arg Gly Gly Tyr Asn Lys

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Claims (6)

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

2. described in claim 1 can antagonism hnRNPU 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.