CN107417772B - Polypeptide HIP-20 capable of antagonizing RNA binding activity of hnRNPU protein and application thereof - Google Patents

Abstract

The invention provides a polypeptide capable of antagonizing the RNA binding activity of hnRNPU protein and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; also relates to an antitumor polypeptide and an application thereof, wherein the antitumor polypeptide comprises a tumor cell killing structural domain and a transmembrane structural domain, and the amino acid sequence of the tumor cell killing structural domain is shown as SEQ ID NO. 1. The transmembrane domain of the anti-tumor polypeptide has no cytotoxicity, but has obvious effects of inhibiting tumor proliferation and migration invasion after being connected with the tumor cell killing domain. The antitumor polypeptide can be used as an antitumor biotherapeutic alone, and is expected to be combined with other treatment modes to inhibit tumors.

Description

Polypeptide HIP-20 capable of antagonizing RNA binding activity of hnRNPU protein and application thereof

Technical Field

The invention relates to the field of tumor targeted therapy, in particular to a polypeptide capable of antagonizing the RNA binding activity of hnRNPU protein and application thereof.

Background

As is well known, tumors are a worldwide medical problem and bring great harm to human health. The conventional tumor treatment medicines are often poor in tissue specificity, damage is caused to normal tissues while tumors are killed, great side effects are caused, and great pain is brought to tumor patients. Therefore, the research and development of tumor targeting polypeptide drugs with high specificity and remarkable curative effect arouse great interest.

The tumor targeting polypeptide refers to a kind of active polypeptide capable of targeting specific molecules related to tumor occurrence and development, and is considered to be the most ideal tumor targeting treatment means at present. Compared with the traditional chemotherapy drugs, the tumor targeting polypeptide has the following advantages: 1. the tissue penetrability is good, and the tumor cells can easily enter the tissue to kill the tumor cells; 2. the biological activity is high, the specificity is strong, the immunogenicity is low and the toxic reaction is relatively weak; 3. the plasma removing speed is high, and accumulation is not easy to generate in vivo; 4. less interaction with other drugs and higher affinity with in vivo targets; 5. is easy for chemical synthesis. Therefore, in recent years, many scholars have made efforts to find short peptides targeting tumor-specific molecules for the purpose of targeting tumors.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RNA binding proteins involved in a variety of pathophysiological processes such as tumorigenesis, viral infection, and apoptosis. Among them, hnRNPU is a phosphorylated protein having the largest molecular weight, and plays an important role in transcription, localization and expression of genes, particularly in the process of apparent inactivation of sex chromosomes. hnRNPU is mostly involved in the regulation of cell functions in the form of DNA/RNA protein complexes, and the scaffold binding region can enhance the functions of transcription factors and regulate the transcription of genes. More and more studies have demonstrated that: the hnRNPU protein is highly expressed in various tumors, and participates in invasion and metastasis of liver cancer and lung cancer by combining with non-coding RNA in vivo. Although there have been many reports of novel antitumor polypeptides, some of which have been put into clinical trials, there have been no reports of antitumor polypeptides against hnRNPU protein.

Therefore, it is necessary to construct a novel polypeptide which can target tumor cells and can efficiently enter cells.

Disclosure of Invention

In order to solve the above problems, the inventors have intensively studied the mechanism of action of hnRNPU protein, and found that hnRNPU promotes transcription of downstream oncogenes or represses transcription of cancer suppressor genes by binding to long non-coding RNA (LOC101927219) through its RGG domain. Blocking the interaction between the RGG structural domain of hnRNPU protein and lncRNA, can effectively suppress the tumor progression.

Based on the research, the invention provides a polypeptide capable of antagonizing the RNA binding activity of hnRNPU protein, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. Experiments prove that the polypeptide can competitively antagonize the combination of cancer-promoting genes hnRNPU and LncRNA, and shows obvious tumor inhibition effect at a cellular level.

The invention also provides application of the polypeptide capable of antagonizing the RNA binding activity of hnRNPU protein in preparation of antitumor drugs.

The invention also provides an anti-tumor polypeptide which comprises a tumor cell killing structural domain and a transmembrane structural domain, wherein the amino acid sequence of the tumor cell killing structural domain is shown as SEQ ID NO. 1.

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

Preferably, the transmembrane domain is linked to the N-terminus of the tumor cell killing domain.

The invention also provides the application of the anti-tumor polypeptide in preparing anti-tumor medicaments.

The invention has the advantages that the transmembrane domain of the anti-tumor polypeptide has no cytotoxicity, but after the transmembrane domain is connected with the RNA binding active peptide segment of hnRNPU, obvious tumor inhibition effect can be observed at the cellular level. The antitumor polypeptide can be used as an antitumor biotherapeutic alone, and is expected to be combined with other treatment modes to inhibit tumors.

Drawings

FIG. 1 is a fluorescent microscope photograph of HeLa cells and SH-SY5Y cells treated with control peptide and HIP-20 for 48 hours;

FIG. 2 is a MTT colorimetric histogram of cells treated with different concentrations of HIP-20 or control peptides;

FIG. 3 is a MTT colorimetric statistical plot of 20. mu. mol/L HIP-20 or control peptide treated cells at various times;

FIG. 4 is a photograph of an experiment for forming a soft agar colony;

FIG. 5 is a statistical graph of the number of cell clones calculated according to FIG. 4;

FIG. 6 is a photograph of a Transwell cell invasion assay;

FIG. 7 is a statistical plot of the migration numbers of tumor cells calculated from FIG. 6;

FIG. 8 is a photograph showing the experiment of inhibiting the angiogenic activity of tumor cells;

FIG. 9 is a statistical plot of the relative angiogenic activity of tumor cells;

FIG. 10 is a photograph showing a Peptide pull down experiment;

FIG. 11 is a photograph of an RNA co-immunoprecipitation experiment (RIP).

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

1. Synthesis of antitumor polypeptides

Synthesizing an anti-tumor polypeptide by a solid phase synthesis method, wherein the anti-tumor polypeptide comprises a tumor cell killing structural domain and a membrane penetrating structural domain, the sequence of the tumor cell killing structural domain is shown as SEQ ID NO. 1, the sequence of the membrane penetrating structural domain is shown as SEQ ID NO. 2, the anti-tumor polypeptide is connected to the N end of the tumor cell killing structural domain, and the obtained sequence is as follows: the amino acid sequence was YGRKKRRQRRR-NMRGGNFRGGAPGNRGGYNK (SEQ ID NO:3), designated HIP-20. For the convenience of research, fluorescein isothiocyanate labeled FITC was attached to the C-terminus of the antitumor polypeptide.

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

a. weighing n equivalents of resin, putting the resin into a reactor, adding DCM (dichloromethane) to swell for half an hour, then pumping off the DCM, adding 2n equivalents of the first amino acid in the sequence, adding 2n equivalents of DIEA (diisopropylethylamine), a proper amount of DMF (dimethylformamide) and DCM (a proper amount is that the resin can be fully stirred), and carrying out nitrogen bubbling reaction for 60 minutes. Then adding about 5n equivalent of methanol, reacting for half an hour, pumping out reaction liquid, and washing with DMF and MEOH;

b. the second amino acid in the sequence (also 2n equivalents), 2n equivalents HBTU (1-hydroxy, benzo, trichloroazol tetramethyl hexafluorophosphate) and DIEA, nitrogen were charged to the reactor and bubbled for half an hour, the liquid was washed off, ninhydrin detected, and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin;

c. adding different amino acids in the sequence in sequence according to the mode of the step b and carrying out various modifications;

d. blowing the resin to dry by using nitrogen, taking the resin out of the reaction column, pouring the resin into a flask, adding a certain amount of cutting fluid (the composition of the cutting fluid and the resin is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water) into the flask, shaking and filtering the resin;

obtaining filtrate, then adding a large amount of ether into the filtrate to separate out a crude product, then centrifuging and cleaning to obtain a crude product of the sequence;

polypeptide purification: purifying the crude product by reversed phase HPLC, connecting the polypeptide to the column packing material by hydrophobic effect, and gradually reducing the ionic strength for elution. The polypeptide ultraviolet absorption is measured by adopting an ultraviolet spectrophotometry to quantify.

Transmembrane and cellular localization assay for HIP-20

Experiments were performed using the human neuroblastoma cell line SH-SY5Y and the human cervical cancer cell line HeLa, respectively. Cells in logarithmic growth phase were seeded on coverslips in 24-well plates at about 8000-. The cells were cultured in 10% fetal Bovine Serum (BSA), high-glucose DMEM medium, and 20. mu. mol/L of the polypeptide was added thereto and maintained for 48 hours. The supernatant was discarded, and 300. mu.l/well of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 20 minutes, and then discarded, and washed twice with 1 XPBS for 5 minutes each. Add 300. mu.l/well of DAPI solution, stain nuclei for 5 minutes at room temperature, wash well 5 times with 1 XPBS for 5 minutes each. The cell membrane was then stained with cell membrane dye at 2. mu. mol/L concentration 200. mu.l/well for 5-10 minutes at room temperature with shaking during staining, followed by 5 washes with 1 XPBS for 5 minutes each. Pure glycerol mounting, cover slip fixed on glass slide. And detecting the positioning of the polypeptide with the fluorescent group in the cell by adopting a laser confocal microscope, and photographing and storing.

The result is shown in figure 1, the polypeptide can effectively enter tumor cells after being added into a cell culture system for 48 hours, and shows extremely strong nuclear localization. Whereas the control peptide was only partially incorporated into the cytoplasm and very little distributed in the nucleus. Therefore, the polypeptide can effectively enter tumor cells and is positioned in the nucleus.

MTT colorimetric assay for detecting inhibition effect of HIP-20 on tumor cell growth

MTT cell viability colorimetric experiments were performed using human neuroblastoma cell line SH-SY5Y, respectively. In a 96-well cell culture plate, 5000 cells were seeded per well, and 8 replicate wells were set for each sample. After 12 hours, HIP-20 and control peptide were added. The IC50 was first detected by administering 0.05, 0.5, 5, 50. mu. mol/L polypeptide. Secondly, according to the concentration of the drug corresponding to IC50, the concentrations of the given polypeptide and the reference polypeptide are respectively 5, 10, 20 and 40 mu mol/L according to IC 50; finally, 20. mu. mol/L of the polypeptide at the specified concentration and the control peptide were administered for 12, 24, 48, and 72 hours, respectively.

After the calibration time was reached, the supernatant was discarded, and the sample wells were washed 3 times with 1 × PBS for 5 minutes each. Then 100. mu.l of dimethyl sulfoxide (DMSO) solution was added to the sample wells. The plates were left to stand at 37 ℃ for 10 minutes. After taking out, the samples in the holes are mixed gently. The absorbance at 570nm was measured with a microplate reader. Data were collected, calculated and counted.

The results are shown in fig. 2 and 3, the viability of the tumor cells treated with the HIP-20 polypeptide is significantly reduced compared to the control polypeptide, and the effect is time-dependent and dose-dependent, indicating that the novel polypeptide can effectively reduce the cell viability of the tumor cells; the effect is evident at a concentration of 20. mu. mol/L for 48 hours. Whereas the control peptide did not show significant growth inhibitory effect.

4. Soft agar colony formation experiment for detecting inhibition effect of HIP-20 on tumor cell proliferation activity

SH-SY5Y and HeLa cells in logarithmic growth phase were taken, digested with 0.25% trypsin and gently blown to make them into single cells for viable cell counting. The number of cells in each well of the six-well plate is controlled to be 1000. Preparing 1.2% and 0.7% low melting point agarose by distilled waterThe solution was autoclaved and maintained at 40 ℃ without coagulation. Adding polypeptide into culture medium at 20 μmol/L concentration, mixing 1.2% agarose and 2 × 1640 culture medium (containing 2 × antibiotic and 20% calf serum) at a ratio of 1:1, adding 3ml mixed solution into six-well plate, cooling and solidifying, and placing CO as bottom layer agar₂And 4, keeping the temperature in the incubator for later use. Mixing 0.7% agarose and 2 XDMEM medium in a sterile test tube according to the proportion of 1:1 to prepare 2ml of upper layer gel, adding 0.2ml of cell suspension into the tube, fully mixing the cell suspension and the upper layer gel, and injecting the cell suspension into a bottom layer plate to form a double agar layer. After the upper agar is solidified, the mixture is placed at 37 ℃ in 5% CO₂Culturing in incubator for 21-28 days until it forms cell colony. 0.5% MTT solution was added to the six-well plate after completion of the culture, stained at 4 ℃ for 2 to 3 hours, and taken out to observe the photograph. The number of colony formations was counted.

The results are shown in FIGS. 4 and 5, and the clone formation of tumor cells treated with HIP-20 polypeptide at a concentration of 20. mu. mol/L is significantly reduced compared to the control polypeptide, indicating that the novel polypeptide can effectively inhibit the proliferation ability of tumor cells.

Transwell experiment for detecting influence of HIP-20 on migration activity of tumor cells

In a 24-well plate, a Transwell chamber (cat. No.: 3422) of Corning corporation was placed, and complete medium containing 15% fetal bovine serum was added to the bottom layer. And the control peptide and HIP-20 were added to the medium at a concentration of 20. mu. mol/L. Single cell suspensions were prepared in serum-free medium and after counting 200. mu.l of cell suspension was added to the upper Transwell chamber at a concentration of 8000- & gt 20000 cells in 200. mu.l of serum-free medium. And the polypeptide is added to the upper chamber at the same concentration as the lower chamber. 5% CO₂And incubated at 37 ℃ for 24 hours. The cell was taken out and fixed in 4% paraformaldehyde for 20 minutes, and then placed in 0.5% crystal violet staining solution and stained at room temperature for 2 hours. Cells in the upper chamber that did not cross the porous membrane were gently removed with a cotton swab and the porous membrane was counted photographically under an inverted microscope.

As shown in FIGS. 6 and 7, the number of tumor cells migrated was significantly reduced in the HIP-20 treated polypeptides compared to the control polypeptides, indicating that the novel polypeptides could effectively inhibit the ability of tumor cells to migrate.

Assay for inhibition of tumor cell angiogenic Activity by HIP-20

The angiogenesis activity of tumor cells can be detected by detecting the growth and the angiogenesis state of SH-SY5Y and HeLa cells in extracellular matrigel; in the following embodiment, SH-SY5Y and HeLa cells were plated in 12-well plates at 70-80% density, 1ml of culture medium was added to each well, and polypeptides and control peptides at 20. mu. mol/L concentration were added; at 37 deg.C, 5% CO₂Culturing in the environment for 48 hours, collecting supernatant in the culture hole, marking and freezing at-80 ℃ for later use; taking out the matrigel from-20 ℃, and rewarming in an ice-water bath to a molten state. Taking a 48-hole cell culture plate, adding 100 mu l of matrigel into each hole to the bottom of each hole, horizontally placing the cell culture plate in an incubator at 37 ℃ for 30 minutes until the matrigel is solidified; preparing HUVEC single cell suspension, counting, adding 8000-10000 cells into each hole of a 48-hole plate, and adding 100 mu l of collected control polypeptide and SH-SY5Y and HeLa cell culture supernatant of HIP-20 treatment group; at 37 deg.C, 5% CO₂Culturing in environment for 6-8 hr, dynamically observing the formation state of HUVEC tubule in real time under an inverted microscope, and taking pictures at appropriate time.

As shown in FIGS. 8 and 9, the polypeptide at a concentration of 20. mu. mol/L significantly inhibited tumor cell angiogenesis compared to the control peptide.

Mechanism of action of HIP-20 in inhibiting tumors

RNA pull-down and RIP detection are adopted to discuss the tumor inhibition mechanism of HIP-20.

HeLa was used to detect the inhibitory effect of the polypeptide on the interaction of LncRNA with hnRNPU protein. Adding polypeptides (0, 5, 10, 20 μmol/L) with different concentrations into cells cultured in a 10cm culture dish, and simultaneously setting a control polypeptide group with 20 μmol/L for 48 hours; 0.25% trypsin digestion, cell collection, 1 × PBS washing, removing supernatant; fully cracking cell masses by using 1ml of RIPA lysate, dividing the lysate into two equal parts, taking 50 mu l of lysate as an Input group, freezing and storing the obtained product at-80 ℃, taking the remaining 450 mu l of lysate as an RIP group, adding 30 mu l of premixed magnetic beads and 1 mu g of LncRNA probe, adding hnRNPU protein antibody and agarose beads into the other group, and placing the obtained product in a refrigerator at 4 ℃ to uniformly mix at 10rpm for overnight; the overnight mixed tube was removed, centrifuged at 3000rpm for 1 minute, the supernatant discarded, 1ml of 1 XPBS was added to resuspend the beads, centrifuged at 3000rpm for 1 minute, the supernatant carefully aspirated, repeated 4-5 times, and the unbound bead fraction washed and discarded. After the last wash, the supernatant was carefully removed and 40. mu.l PBS was added to resuspend the beads; adding 10 μ l of 5 XSDSSPAGE loading buffer, mixing, and performing water bath at 95 ℃ for 10 minutes to dissociate protein and RNA on the beads; the eluted nucleic acid components are subjected to PCR amplification using specific primers.

12% SDS-PAGE gel was prepared, followed by electrophoresis, by cutting a band of hnRNPU protein (120kDa) according to molecular weight, blocking with a membrane, incubation with the corresponding antibody, and exposure. The inhibitory effect of the polypeptides at different concentrations on the interaction of the LncRNA and the hnRNPU protein is judged according to the brightness of the band. Preparing 1.5% agarose gel, removing PCR products with the same volume for electrophoretic identification, and performing semi-quantitative comparison according to the brightness of an electrophoretic band.

As shown in FIGS. 10 and 11, the results of western blot analysis showed that the loading amount of proteins was equal in each group in the Input group, and that in the RNA pull-down group, the hnRNPU proteins bound to the LncRNA probe decreased gradually as the concentration of HOP-20 increased, indicating that the HIP-20 polypeptide inhibited the binding of LncRNA to hnRNPU proteins.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> affiliated cooperation hospital of college of Tongji medical college of Huazhong university of science and technology

<120> polypeptide HIP-20 capable of antagonizing RNA binding activity of hnRNPU protein and application thereof

<130>1

<160>3

<170>PatentIn version 3.5

<210>1

<211>20

<212>PRT

<213> Artificial sequence

<400>1

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

1 5 10 15

Gly Tyr Asn Lys

20

<210>2

<211>11

<212>PRT

<213> Artificial sequence

<400>2

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210>3

<211>31

<212>PRT

<213> Artificial sequence

<400>3

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Asn Met Arg Gly Gly

1 5 10 15

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

20 25 30

Claims (6)

1. A polypeptide capable of antagonizing the RNA binding activity of hnRNPU protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. Use of the polypeptide of claim 1 for antagonizing RNA binding activity of hnRNPU protein in the preparation of drugs for treating neuroblastoma and cervical cancer.

3. An anti-tumor polypeptide is characterized by comprising a tumor cell killing structural domain and a transmembrane structural domain, wherein the amino acid sequence of the tumor cell killing structural domain is shown as SEQ ID NO. 1.

4. The anti-tumor polypeptide of claim 3, wherein the amino acid sequence of the transmembrane domain is represented by SEQ ID NO 2.

5. The anti-tumor polypeptide of claim 3 or 4, wherein the transmembrane domain is linked to the N-terminus of the tumor cell killing domain.

6. Use of the anti-tumor polypeptide of any one of claims 3-5 for the preparation of a medicament for treating human neuroblastoma and a medicament for treating human cervical cancer.

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