CN110317243B

CN110317243B - RAGE (receptor for activating receptor) antagonistic polypeptide and application thereof

Info

Publication number: CN110317243B
Application number: CN201910525765.XA
Authority: CN
Inventors: 黄来强; 代小勇; 蒋盛威; 刘可为; 邓婷; 王丽君
Original assignee: Shenzhen Graduate School Tsinghua University
Current assignee: Shenzhen Graduate School Tsinghua University
Priority date: 2019-06-18
Filing date: 2019-06-18
Publication date: 2021-03-30
Anticipated expiration: 2039-06-18
Also published as: CN110317243A

Abstract

The invention belongs to the field of biotechnology and biomedicine, and particularly relates to a RAGE antagonist polypeptide and application thereof. The RAGE antagonistic polypeptide obtained by screening by taking RAGE as a target through a phage display technology can be specifically combined with the RAGE to block a signal path thereof, so that the proliferation of cancer cells is inhibited, an effective small-molecule drug is provided for RAGE-mediated cancers, and the RAGE antagonistic polypeptide can be widely applied to the fields of medicine and biology.

Description

RAGE (receptor for activating receptor) antagonistic polypeptide and application thereof

Technical Field

The invention belongs to the field of biotechnology and biomedicine, and particularly relates to a RAGE antagonist polypeptide and application thereof.

Background

Breast cancer (Breast cancer) refers to malignant tumor occurring in mammary gland epithelial tissue, 4-6% of Breast cancers are metastatic Breast cancers when diagnosed, 30-40% of patients receiving adjuvant therapy can develop metastatic Breast cancers at early stage, and the 5-year survival rate of the patients is about 20%. However, the traditional treatment modes such as surgery, radiotherapy and chemotherapy have poor specificity and specificity of action, inevitably generate killing effect on normal cells and tissues and bring great side effect to patients. Therefore, the search for efficient and specific targeted drugs can greatly improve the treatment effect of breast cancer.

The targeted therapy of breast cancer molecules refers to the treatment of signal pathways related to the occurrence and development of breast cancer and related expression products of oncogenes of the breast cancer. Receptor for advanced glycation end products (RAGE) is a multi-ligand Receptor that is one member of the immunoglobulin superfamily. RAGE was first discovered in the study of diabetes and was named receptor for Advanced glycation end products (RAGE) because it binds to AGEs, the major causative agent of diabetes, causing damage to tissue. The RAGE structure is divided into an N end and a C end, the N end is an extracellular section and is used for identifying different ligands and comprises 1V area and 2C areas, the main part of the V area is used for being combined with the ligands, and the C area is used for stabilizing the combination of receptors and the ligands; the middle comprises a transmembrane segment; the C-terminal is an intracellular segment which mainly participates in signal transduction pathways. RAGE is distributed very widely in mammalian tissues, such as lymphocytes, endothelial cells, monocytes, macrophages, smooth muscle cells, mesangial cells, neurons, etc. RAGE is expressed at low levels in various physiological tissues, in addition to being highly expressed in lung tissues under normal physiological conditions. In many inflammatory or chronic diseases, such as diabetes, rheumatoid arthritis, trauma, Alzheimer's disease and other pathological environments and growth and development stages, RAGE expression and ligands on the cell surface are obviously enhanced. RAGE is also overexpressed in many types of cancers, such as breast, stomach, prostate, colon, rectum, pancreas, lung, melanoma, etc., and is closely related to proliferation, motility, invasive metastatic capacity of tumor cells, and clinical stage, prognosis, etc. of tumors. Recent studies show that the down-regulation of RAGE expression in different subtype breast cancer cell lines has a profound effect on the proliferation of cancer cells, and the siRNA specifically targeting RAGE can down-regulate RAGE expression level of breast cancer cells and can effectively reduce the proliferation of breast cancer in vivo. However, the siRNA treatment has the obvious defect that the siRNA entering the human body is easily degraded by the body, thereby greatly reducing the action time. The polypeptide medicament has higher targeting property, long action time and small toxic and side effects. The RAGE is taken as a therapeutic target, and the RAGE can be taken as a new strategy for treating the breast cancer.

Disclosure of Invention

In order to solve the above technical problems, the present invention aims to provide a novel RAGE antagonist polypeptide, derivatives thereof and uses thereof.

The technical scheme adopted by the invention is as follows:

a RAGE antagonist polypeptide comprising the amino acid sequence HPMSAPR (SEQ No.: 1).

Further, the RAGE antagonist polypeptide is obtained by phage display random peptide library screening. Phage Display Technology (Phage Display Technology) is a screening Technology for specific polypeptides or proteins, which can Display the polypeptide encoded by a target gene on the surface of Phage in the form of fusion protein, and the displayed polypeptides or proteins can maintain relatively independent spatial structure and biological activity, so that the polypeptide ligands of various target molecules (such as antibody, enzyme and cell surface receptor, etc.) can be rapidly identified by in vitro affinity panning procedures.

Further, the RAGE antagonist polypeptide may be a fragment of a polypeptide obtained from a native protein or may be obtained using well known methods of peptide synthesis.

Further, the RAGE antagonist polypeptide specifically binds to RAGE.

The present invention also provides a derivative of the RAGE antagonist polypeptide as a product of a modification of the amino acid side chain moiety of the RAGE antagonist polypeptide, the amino terminus or the carboxy terminus of a fragment of the RAGE antagonist polypeptide, or a product of a ligation of the RAGE antagonist polypeptide to a tag for polypeptide or protein detection or purification.

Further, the modification comprises amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification; the label is His₆GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.

Further, derivatives of the RAGE antagonist polypeptide can be chemically synthesized using automated polypeptide synthesizers, using methods well known in the art; deducing a nucleotide sequence from the short peptide sequence, and cloning the nucleotide sequence into a vector for biosynthesis; it can also be extracted and purified in large quantities from existing organisms.

The invention also provides a nucleotide sequence encoding the RAGE antagonist polypeptide.

The invention also provides an expression vector which contains the nucleotide sequence.

The invention also provides a host cell which contains the expression vector.

The invention also provides a method of producing a protein capable of binding RAGE comprising expressing a vector comprising the amino acid sequence of hpmaspr (SEQ No.:1) in a host cell.

The present invention also provides a pharmaceutical composition comprising the RAGE antagonist polypeptide or a derivative of the RAGE antagonist polypeptide.

Further, the pharmaceutical composition also includes an agent capable of killing cancer cells; preferably, the agent is at least one of a chemical drug, a nano-drug, a biological drug, a radiopharmaceutical, a photothermal therapy or a photodynamic therapy drug capable of killing cancer cells; preferably, the agent is a chemical or biological drug.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; preferably, the carrier is at least one of a sustained release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, or a lubricant.

The invention also provides the use of a RAGE antagonist polypeptide as described above for the preparation of a composition for the targeted recognition of RAGE.

The invention also provides the use of the RAGE antagonist polypeptide or a derivative of the RAGE antagonist polypeptide or the pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of cancer.

Further, the cancer is a RAGE overexpressing cancer.

Further, the cancer is breast cancer, lung cancer, prostate cancer, stomach cancer, rectal cancer, colon cancer, and pancreatic cancer.

Further, the cancer is triple negative breast cancer.

Further, the medicament also comprises a pharmaceutically acceptable carrier.

Further, the medicine can be prepared into tablets, granules, capsules, oral liquid or injections.

The invention also provides the application of the derivative of the RAGE antagonist polypeptide in the preparation of a medicament for preventing and/or treating cancer.

The invention has the beneficial effects that:

the invention provides a RAGE antagonistic polypeptide which is obtained by screening by taking RAGE as a target; the compound can be combined with RAGE to block a mediated signal path and inhibit the proliferation of cancer cells, can be used for preparing and/or treating RAGE over-expressed cancers, such as breast cancer, lung cancer, prostate cancer, gastric cancer, rectal cancer, colon cancer, pancreatic cancer and the like, can be widely applied in the fields of medicine and biology, and can generate great social and economic benefits.

Drawings

FIG. 1 shows QPCR detection of RAGE expression levels in different cells;

FIG. 2 illustrates specific binding of a confocal detection RAGE antagonist polypeptide to RAGE;

FIG. 3 shows MTT assay to determine the effect of RAGE antagonist polypeptides on the proliferative capacity of various cancer cells;

FIG. 4 shows MTT assay to detect the effect of RAGE antagonist polypeptides on the proliferative capacity of MDA-MB-231 cells at various times;

FIG. 5 shows EDU staining to detect the effect of RAGE antagonist polypeptides on the proliferative capacity of MDA-MB-231 cells.

Detailed Description

The technical solutions of the present invention are further described below with reference to the drawings and specific examples, but the present invention is not limited to these specific embodiments. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.

Example 1QPCR detection of RAGE protein expression levels in different cancer cells

Taking normal breast cells MCF-10A, estrogen positive (ER +) cells MCF-7 and triple negative cells MDA-MB-231 cells.

RNA extraction: in the case of a 6cm dish, when the cell growth state is good and the confluency of the cells reaches 80 to 90%, the cell culture solution is discarded, and 1 XPBS is added to wash the cells to remove the remaining culture solution. Then, 300-500. mu.L of Trizol (the amount of Trizol added is determined according to the cell density) is added into a 6cm culture dish, and the pipetting is repeated until the cells are ruptured and no precipitate is evident in the lysate. Place it in a 1.5mL EP tube (note use of RNase free tip and EP tube, and preferably wear a mask); placing on ice for 5min, adding chloroform according to the proportion of Trizol to chloroform of 5:1, and shaking vigorously for 15s, wherein chloroform can effectively separate an organic phase from an inorganic phase, and RNA enters a water phase; standing on ice for 2-3min, centrifuging at 4 deg.C for 15min at 12000 g; carefully adding the upper aqueous phase into a new EP tube, adding isopropanol with the same volume to precipitate RNA, and placing on ice for 10 min; centrifuging at 4 ℃ for 10min at 12000g for 10min to remove supernatant, cleaning RNA precipitate with 75% absolute ethyl alcohol, blowing and sucking with a gun head without RNase, centrifuging at 4 ℃ for 5min at 7500g, removing supernatant, placing in a safety cabinet, opening, air drying for 3-4min, determining the amount of DEPC water to be used according to the amount of precipitate, mixing uniformly, measuring the concentration of RNA, and performing agarose gel electrophoresis to verify the quality of RNA. Finally storing in a refrigerator at-80 ℃ for later use.

First strand cDNA Synthesis: the ingredients were first mixed in an EP tube. The total RNA template usage amount is generally 1-5 μ g, the above samples are mixed, centrifuged, placed in a 65 deg.C water bath for 5min, and then rapidly placed on ice for 2 min. Adding the primers, mixing, placing in a water bath at 42 ℃ for 1 hour, then placing in a 70 ℃ for heat preservation for 15min, and finally obtaining the cDNA which can be used for the next PCR experiment.

And (3) adding the detection index of the sample to be detected and the internal reference (GAPDH) thereof according to a reaction system, wherein each sample needs to be provided with three multiple holes. During operation, the cDNA, SYBR Green1MIX and water can be mixed uniformly, and each sample is ensured to be subjected to three-hole and two-detection indexes, so that 6 parts of the total amount of the cDNA, the SYBR Green1MIX and the water are required to be prepared on the basis of the original dosage in the system. And similarly, uniformly mixing the upstream primer and the downstream primer according to the dosage requirement, finally sequentially adding a system with 20 mu L of each hole of the two mixed solutions into the PCR eight-connected tube, centrifuging and loading on the machine. The cycle is then 35-40, and dissolution profile is performed. And then calculating the expression quantity of the target to be detected according to the measured CT value.

Primer information for RAGE:

Fw 5’-TGCTGTACTTTGGAAGCGAAC-3’(SEQ ID No.：2)

Rv 5’-CCGAGGGTTGACATGGAACTG-3’(SEQ ID No.：3)

GAPHD primer information:

Fw 5’-TGTGGGCATCAATGGATTTGG-3’(SEQ ID No.：4)

Rv 5’-ACACCATGTATTCCGGGTCAAT-3’(SEQ ID No.：5)

as shown in FIG. 1, it can be seen that the RAGE receptor is poorly expressed in normal mammary MCF-10A, estrogen positive (ER +) MCF-7, and highly expressed in triple negative MDA-MB-231 cells.

Example 2 screening, Synthesis and identification of RAGE antagonist Polypeptides

1. Screening for RAGE antagonist polypeptides

(1) Establishment of 293T cell line permanently highly expressing RAGE: 293T-RAGE^+/+/LRH

Selecting human 293T cell with vigorous growth, and culturing at 5X 10 times the day before transfection⁵One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

② the second day, with one culture well of 6-well plate as a unit, diluting 3. mu.g RAGE expression plasmid with 200. mu.L opti-MEM culture medium, diluting 6. mu.L liposome Lipofectamine2000 with 200. mu.L opti-MEM culture medium, mixing gently, standing at room temperature for 5 minutes;

③ mixing the two tube dilutions gently, standing for 20 minutes at room temperature, and then adding 600 μ L of opti-MEM culture medium gently into the mixed dilutions;

rinsing the cells to be transfected with PBS slightly once, then adding the mixed diluent into the culture holes slightly, and culturing in a carbon dioxide incubator;

fifthly, after culturing for 4-6 hours, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;

sixthly, selecting a culture medium containing 1 mu g/mL puromycin after 48 hours for screening; obtaining the 293T cell line which stably expresses RAGE after the cells are not dead any more.

Seventhly, extracting total RNA by using TRIzol, quantifying 2 mug of RNA for reverse transcription (a reverse transcription kit, purchased from Promega corporation), and performing qPCR by using a specific primer sequence.

The sequence of the specific primer is as follows:

Fw 5’-TGCTGTACTTTGGAAGCGAAC-3’(SEQ ID No.:2)

Rv 5’-CCGAGGGTTGACATGGAACTG-3’(SEQ ID No.:3)

(viii) comparing with transfected pSM2c-Hu-scramble RNA, detecting the high expression level of RAGE, and naming as: 293T-RAGE^+/+LRH, i.e., can be used for positive phage selection.

(2) Panning, amplification, purification, sequencing and synthesis of RAGE antagonist polypeptides.

Preparation of ER2738 host bacterial liquid: performing aseptic technique operation, namely taking 200 mu L of LB-Tet liquid culture medium in a 1.5mL sterile centrifuge tube, taking 0.2 mu L of bacterial liquid from the glycerol frozen product of E.coli ER2738, fully and uniformly mixing the bacterial liquid with the glycerol frozen product, completely absorbing and coating the bacterial liquid on an LB-Tet plate, marking the plate, standing the plate at room temperature for 3min, and then placing the plate in a 37 ℃ constant temperature incubator for inversion overnight culture. Observing the next day, sealing with sealing film after clone grows out, and storing at 4 deg.C in dark for use. Single colonies were picked aseptically with a sterile pipette tip and placed into 10mL sterile centrifuge tubes pre-filled with 3mL LB-Tet broth, labeled and shake-cultured overnight on a constant temperature shaker at 37 ℃ and 300 rpm/min. The next day, the bacterial amplification solution was stored at 4 ℃ for future use. Taking 10mL of a sterilized centrifuge tube, adding 3mL of LB-Tet liquid culture medium in a sterile operation manner, inoculating 30 mu L of overnight-cultured bacteria, carrying out shake culture at constant temperature of 37 ℃ and 300rpm/min for 2-3 h, wherein the bacteria are in an exponential growth phase and are in a fog shape (OD) by visual observation₆₀₀～0.5)。

Elutriation of RAGE antagonist peptides: high expression RAGE cells are administered as 10⁵The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance²In a culture dish, culturing the cells to 80-9% of cell density by a conventional methodAt 0%, 1 μ L of each elution run was used for panning (while using a cell line that does not express RAGE as a blank control), the remainder was added to 20mL of LB medium for amplification, followed by purification and final re-measurement of the amplified titer, the amplificate was stored at 4 ℃ for a short period and an equal number of steps was used for the next panning, and the remainder was stored at-20 ℃ in 50% glycerol.

Measuring the titer of the phage: 4 sterilized 10mL centrifuge tubes were prepared for each phage dilution, and Top agar (agar Top) was melted in a microwave oven, 3mL Top agar was added to each tube, and a water bath was run at 45 ℃ until needed. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. Will OD₆₀₀Coli ER2738 E.coli 0.5 was aliquoted at phage dilution 200. mu.L/tube and stored at 4 ℃ for future use. Taking 4 sterilized 1.5mL centrifuge tubes, respectively containing 100 μ L, 90 μ L LB-Tet culture medium, sucking 1 μ L of bacteriophage to be tested into 100 μ L LB-Tet culture medium, diluting according to 10 times gradient, respectively marking as 10^-1、10^-2、10^-3、10^-4And each dilution is mixed evenly by gentle oscillation and then is centrifuged instantly. Mix 10 μ L of each dilution of phage to be titrated with 200 μ L of e.coli ER2738, mix by gentle shaking, centrifuge instantaneously, incubate for 5min at room temperature. Quickly adding the mixed bacterial liquid into top agar, quickly shaking and uniformly mixing, immediately pouring into a preheated LB/IPTG/Xgal plate, uniformly flattening, cooling at room temperature for 5min, and inversely culturing the plate in a constant-temperature incubator at 37 ℃ overnight.

Amplification and purification of eluted phage: taking a 250mL conical flask, adding the overnight cultured ER2738 host bacterial liquid into 20mL LB liquid culture medium according to the proportion of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; then adding the phage liquid to be amplified into an erlenmeyer flask, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 4.5 h; the culture was transferred to a 50mL centrifuge tube and centrifuged at 10,000rpm at 4 ℃ for 10 min. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm at 4 ℃ for 10 min; transferring 80% of the supernatant into another clean centrifuge tube, adding 1/4 volume of PEG/NaCl, reversing, mixing uniformly, and precipitating at 4 ℃ overnight; the next day, the pellet was centrifuged at 12,000rpm for 20min at 4 ℃. Carefully sucking the supernatant with a clean gun head, centrifuging at 4 deg.C and 12,000rpm for 1min, and removing the residual supernatant; the pellet was then resuspended in 1mL TBS and gently pipetted 100 times. Then transferring the suspension into a 2mL centrifuge tube, and centrifuging at 4 ℃ and 10,000rpm for 5min to remove residual cells; adding 1/4 volume of PEG/NaCl to the supernatant, and incubating on ice for 60min for reprecipitation; taking out the centrifuge tube, centrifuging at 4 deg.C and 12,000rpm for 20min, and removing supernatant; the pellet was resuspended in 200. mu.L TBS and centrifuged at 10,000rpm for 1min at 4 ℃. The supernatant was transferred to another centrifuge tube. Short-term storage at 4 deg.C, or long-term storage at-20 deg.C with 50% glycerol. The amplification of the monoclonal phage comprises the steps of adding overnight cultured ER2738 host bacterial liquid into 2mL of LB liquid culture medium according to the proportion of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; selecting a plate with less than 100 plaques from the fourth round of titer plates by using a sterilizing toothpick, picking well-separated blue plaques, adding the blue plaques into a culture tube, and carrying out violent shake culture at 37 ℃ and 250r/min for 4.5 h; the culture was then transferred to fresh centrifuge tubes and centrifuged at 10,000rpm for 30sec at 4 ℃. Transferring the supernatant into a fresh tube, and centrifuging once again; 80% of the supernatant was transferred to fresh centrifuge tubes and stored at 4 ℃ or stored with 50% glycerol for a long period at-20 ℃.

Identifying M13 bacteriophage ssDNA by agarose gel electrophoresis: horizontally placing a gel forming mold, placing the selected comb, and reserving a space of 1mm between the bottom of the comb and the mold; weighing 1g of agarose for DNA electrophoresis, putting the agarose into a 250mL Erlenmeyer flask, adding 100mL of 1 XTAE buffer solution, uniformly mixing, putting the flask into a microwave oven, heating and boiling until the agarose is completely dissolved; and (3) closing the induction cooker, taking out the triangular flask, cooling the triangular flask to room temperature (which can be tolerated by holding the flask by hand), adding 5 mu L of ethidium bromide, and pouring the gel solution into a rubber plate paving plate after uniformly mixing. The rubber plate used in the experiment needs about 100mL of rubber solution; after the gel is completely solidified at room temperature and takes about 30 minutes, pulling out the comb teeth, and putting the rubber plate into an electrophoresis tank; adding 1 XTAE buffer solution into the electrophoresis tank, preferably 2mm higher than the surface of the gel; diluting the sample with a Loading buffer solution (Loading buffer), and then adding the sample into a gel plate, wherein a suction head of a sample loader is just placed in a gel spot sample hole, so that the gel cannot be punctured, and the sample is prevented from overflowing out of the hole; switching on a power supply, adjusting the voltage to 50V, performing electrophoresis for 90min, taking out the gel plate, and observing the result under an ultraviolet lamp.

Sequencing and sequence analysis of ssDNA: the extracted M13 phage ssDNA was sent to Shanghai Yingji Biotechnology Ltd for DNA sequencing. Sequencing was followed by sequence analysis using Bioedit software. From the results of the analysis, the sample sequence was HPMSAPR (SEQ NO: 1), and finally the RAGE antagonist polypeptide was synthesized by Shanghai Yao Bio Inc.

2. Synthesis and identification of RAGE antagonist polypeptides

The polypeptide was synthesized by Fmoc solid phase synthesis (synthesized by Shanghai Qiaozao Bio Inc.) using a CS936 polypeptide synthesizer (CSBio Inc., USA), and the synthesis process included the following steps:

(1) deprotection: removing the protecting group of the amino group by using piperidine (piperidine, Shanghai purple reagent factory);

(2) activation and crosslinking: the carboxyl of the next amino acid is activated and dissolved by activator HBTU (HCTU/HITU) + NMM, and the activated monomer reacts with free amino to form peptide bond;

(3) and (3) circulation: (1) and (2) the two steps of reaction are repeatedly circulated until the synthesis of the whole peptide chain is finished;

(4) elution and deprotection: eluting the column with different resin-removing solvents according to the residues contained in the peptide chain, wherein the protecting groups are eluted and deprotected by a deprotection agent (TFA);

(5) the synthesized short peptide is purified by a Varian Prostar210 purification column (VARIAN, USA), and a UV-Vis-detector which is Varian Prostar345 (VARIAN, USA) is adopted in the purification process;

(6) the purity is verified to be more than 98% by adopting System Gold HPLC (Beckman company in America);

(7) the molecular weight of the synthesized short peptide was measured by Thermo Finnigan LCQ decap XPplus (Thermo Co., USA).

Example 3

Specific binding of RAGE antagonist polypeptides to RAGE

FITC labeling of RAGE antagonist Polypeptides (Shanghai Qiaozhizoe Biopsis Co., Ltd.)The focused plate was inoculated with MDA-MB-231 cells, washed three times with PBS, fixed in paraformaldehyde for 15min, washed 3 times with PBS, blocked with BSA for 1.5h, incubated overnight with RAGE antibody, and then treated with Alexa

594 linked anti-rabbit fluorescent secondary antibodies were incubated for 1 hour at room temperature, then incubated with FITC-labeled RAGE antagonist polypeptide at a final concentration of 100. mu. mol/mL for 2 hours at room temperature, washed three times with PBST, stained with DAPI nuclei, and observed by confocal laser scanning microscopy (Seiss, Germany), with the results shown in FIG. 2.

Wherein, fig. 2A is a fluorescent confocal microscope picture of DAPI nuclear staining (the original fluorescent color picture shows blue);

FIG. 2B shows Alexa after incubation of MDA-MB-231 cells with RAGE antibody

594 linked to an anti-rabbit fluorescent secondary antibody (shown in red in the original fluorescent color picture);

FIG. 2C is a confocal microscope image of fluorescence labeled with FITC labeled RAGE antagonist polypeptide (shown as green in the original fluorescence color);

FIG. 2D is a merged image of FIGS. 2A, 2B and 2C (shown as purple in the original fluorescence color).

It can be seen that the FITC-labeled RAGE antagonist polypeptide co-localizes with an antibody that specifically binds RAGE, indicating that the RAGE antagonist polypeptide is capable of specifically binding RAGE.

Example 4

Effect of RAGE antagonist Polypeptides on the proliferative Capacity of different mammary cells

Firstly, the normal mammary cell MCF-10A, the estrogen positive (ER +) cell MCF-7 and the triple negative cell MDA-MB-231 are respectively divided into 5 x 10 cells³Inoculating each well into a 96-well cell culture plate, culturing for 24h with the culture medium volume of 200 mu L per well, and then starving overnight;

② adding RAGE antagonistic polypeptide with different concentration gradients (100 μ M, 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M) to culture for 48 hours;

③ adding 20 mu L of MTT working solution into each hole, and continuously putting into a carbon dioxide incubator to culture for 4 hours;

and fourthly, abandoning the supernatant in the culture plate, adding 150 mu L DMSO (dimethyl sulfoxide), shaking for 10 minutes, selecting 490nm wavelength on a microplate reader for detection, and drawing a growth curve of the cells.

FIG. 3 is a diagram of MTT assay for detecting the effect of RAGE antagonist polypeptide on the proliferative capacity of different mammary cells, and it can be seen that RAGE antagonist polypeptide can significantly inhibit the proliferation of RAGE high-expressing cells MDA-MB-231, while it has no significant inhibitory effect on the proliferation of RAGE receptor low-expressing cells MCF-10A and MCF-7.

Example 5

Effect of RAGE antagonist Polypeptides on the proliferative Capacity of Breast cancer cells at different times

(ii) triple negative MDA-MB-231 cells were reduced to 5 x 10³Inoculating each well into a 96-well cell culture plate, culturing for 24h with the culture medium volume of 200 mu L per well, and then starving overnight;

② RAGE antagonistic polypeptide with different concentration gradients (100 μ M, 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M) is added to be cultured for 24, 48 and 72 hours respectively;

FIG. 4 is a graph showing the effect of the MTT assay on the proliferation potency of RAGE antagonists at various times in breast cancer cells, and it can be seen that the RAGE antagonists significantly inhibited cell proliferation at 24, 48, and 72 hours, and particularly at 48 hours, the inhibition was most pronounced.

Example 6

EDU staining method for detecting influence of RAGE antagonistic polypeptide on MDA-MB-231 cell proliferation

(1) Cell culture: taking cells in logarithmic growth phase at 4X 10 per well³～1×10⁵Inoculating the cells in a 96-well plate, and culturing to a normal growth stage;

(2) and (3) drug treatment: adding 10 mu M of short peptide for 48 hours;

(3) EDU marking:

1) the cells were cultured in a cell culture medium at 1000: 1 (reagent A) to prepare an appropriate amount of 50. mu.M EdU medium;

2) adding 100 mu L of 50 mu M EdU culture medium into each hole, incubating for 2 hours, and removing the culture medium;

3) the cells were washed 1-2 times with PBS for 5 minutes each time.

(4) Immobilization of cells

1) Adding 50 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30 min, and discarding the fixing solution;

2) adding 50 mu L of 2mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, removing the glycine solution;

3) adding 100 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS;

4) (boost) Add 100. mu.L of osmotic agent (0.5% TritonX-100 in PBS) per well and incubate for 10min on a decolorized shaker; PBS wash 1 times, 5 minutes.

(5) Apollo staining

1) Add 100. mu.L of 1X per well

Incubating the dyeing reaction liquid for 30 minutes in a dark place at room temperature by using a decoloring shaker, and then discarding the dyeing reaction liquid;

2) adding 100 mu L of penetrating agent (PBS of 0.5 percent TritonX-100) to decolor and wash by a shaking table for 2-3 times, each time for 10 minutes, and discarding the penetrating agent;

3) (reinforcing) adding 100 mu L of methanol into each hole for cleaning for 1-2 times, and each time for 5 minutes; PBS wash was performed 1 time for 5 minutes each.

(6) DNA staining

1) Deionized water was added according to a 100: 1, preparing a proper amount of 1X Hoechst33342 reaction solution, and storing in a dark place;

2) adding 100 mu L of 1X Hoechst33342 reaction solution into each hole, incubating for 30 minutes in a light-proof, room temperature and decolorizing shaker, and then discarding the dyeing reaction solution;

3) adding 100 mu L PBS into each hole for washing for 1-3 times;

4) add 100. mu.L PBS per well and store until use.

FIG. 5A is an EDU staining fluorescence assay: from left to right: DAPI nuclear staining (shown as blue in the original fluorescence color image); EDU-labeled DNA (shown green in the original fluorescence color); combined previous DAPI and EDU staining (purple in the original fluorescence color)

FIG. 5B is a statistical chart of EDU staining

FIG. 5 shows that the short peptide can significantly inhibit the synthesis of cell DNA at a concentration of 10. mu.M for 48 hours, thereby inhibiting the proliferation of cells.

It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

SEQUENCE LISTING

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<120> RAGE antagonistic polypeptide and application thereof

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Claims

Use of a RAGE antagonist polypeptide in the manufacture of a medicament for the prevention and/or treatment of a cancer, wherein said cancer is breast cancer and said RAGE antagonist polypeptide has a sequence as set forth in SEQ No.: 1.
2. The use of claim 1, wherein the RAGE antagonist polypeptide specifically binds to RAGE.