CN106317219A - PAQR3 polypeptide fragment, medicinal composition thereof, and use of PAQR3 polypeptide fragment or medicinal composition - Google Patents

Abstract

The invention relates to a PAQR3 polypeptide fragment, a medicinal composition thereof, and a use of the PAQR3 polypeptide fragment or the medicinal composition. The invention concretely relates to a separated amino acid sequence. The amino acid sequence contains a fragment in SEQ ID NO:1, wherein the fragment is positioned between the first position and the 70th position in the SEQ ID NO:1. The invention also relates to a coding sequence of the amino acid sequence, a nucleic acid construct containing the coding sequence, the medicinal composition containing the amino acid sequence, and a pharmaceutical use of the amino acid sequence.

Description

PAQR3 polypeptide fragment, its pharmaceutical composition and use

technical field

The present invention relates to PAQR3 polypeptide fragment, its pharmaceutical composition and its use.

Background technique

China is a high-incidence area of cancer, and its morbidity and mortality are higher than the world average [Ferlay J, ShinHR, Bray F, Forman D, Mathers C and Parkin DM, (2010) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int J Cancer 127(12):2893-917]. Cancer is a complex, multistep disease of unknown etiology. Studies in the molecular biology of cancer have proved that the activation of PI3K/Akt and MAPK/Erk signaling pathways is conducive to the growth, proliferation and metastasis of cancer cells [AnS, Yang Y, Ward R, Liu Y, Guo XX and Xu TR, (2015 ) Raf-interactome in tuning the complexity and diversity of Raf function. FEBS J 282(1):32-53; Pal I, (2012) Mandal M. PI3K and Akt as molecular targets for cancer therapy: current clinical outcomes, Acta Pharmacol Sin 33 (12):1441-58].

The applicant's previous results proved that PAQR3, a protein in the PAQR (Progestin and AdipoQ Receptor, progesterone and adiponectin receptor) family, can bind to the catalytic subunit p110 of PI3K and Raf kinase, thereby inhibiting PI3K/Akt and MAPK/Erk signaling Pathways [Feng, L., Xie, X., Ding, Q., Luo, X., He, J., Fan, F., Liu, W., Wang, Z. and Chen, Y., (2007) Spatial regulation of Raf kinase signaling by RKTG, Proceedings of the National Academy of Sciences of the United States of America 104, 14348-53; Wang, X., Wang, L., Zhu, L., Pan, Y., Xiao, F. , Liu, W., Wang, Z., Guo, F., Liu, Y., Thomas, W.G. and Chen, Y., (2013) PAQR3 modulates insulin signaling by shunting phosphoinositide3-kinase p110a to the Golgi apparatus, Diabetes 62( 2):444-56].

Our recent study found that the important functional region of PAQR3 to block the combination of p110α and p85, and the combination of Raf and MEK is at the N-terminus of PAQR3 protein. According to the binding sites of PAQR3 and p110 and Raf, we designed and synthesized the N-terminal fragment containing PAQR3 protein. Studies have shown that this fragment can effectively block the binding of p110α and p85, Raf-1 and MEK, thereby inhibiting these two The phosphorylation levels of the downstream proteins Akt and Erk in the signaling pathway can inhibit the activation of these two pathways in gastric cancer cells, and significantly inhibit the growth and metastasis of tumor cells.

Contents of the invention

The present invention provides an isolated amino acid sequence, the isolated amino acid sequence contains a fragment of SEQ ID NO: 1, wherein the fragment is located between the 1st and 70th positions of SEQ ID NO: 1, and has a length of at least 40 amino acid residues .

In a specific embodiment, the fragment is at least 45 amino acid residues, preferably at least 50 amino acid residues in length.

In a specific embodiment, the fragment contains amino acid residues 6 to 55 of SEQ ID NO:1.

In a specific embodiment, the fragment is located between amino acid residues 3 to 65 of SEQ ID NO:1.

In a specific embodiment, the fragment is located between amino acid residues 5 to 60 of SEQ ID NO:1.

In a specific embodiment, the isolated amino acid sequence consists of said fragment of SEQ ID NO:1.

In a specific embodiment, the isolated amino acid sequence also contains a peptide that facilitates membrane penetration.

In a specific embodiment, the peptide that promotes membrane penetration is selected from the group consisting of: RQIKIWFQNRRMKWKK (SEQ ID NO: 10), YGRKKRRQRRR (SEQ ID NO: 11), KQAIPVAK-amide (SEQ ID NO: 12), RRRRNRTRRNRRRVR-amide (SEQ ID NO: 13), oligoarginine (R ₉ -R ₁₂ ), KLTRAQRRAAARKNKRNTRGC (SEQ ID NO: 14), ALWKTLLKKVLKAPKKKRKVC (SEQ ID NO: 15), RKKRRQRRR (SEQ ID NO: 16), DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 17), GWTLNSAGYLLGKINLKALAALAKKIL-amide (SEQ ID NO: 18), AGYLLGKINLKALAALAKKIL-amide (SEQ ID NO: 19), YTAIAWVKAFIRKLRK-amide (SEQ ID NO: 20) and the like.

In a specific embodiment, said isolated amino acid sequence consists of a peptide that facilitates membrane penetration and said fragment of SEQ ID NO:1.

In a specific embodiment, the isolated amino acid sequence is shown as SEQ ID NO:3.

The present invention also provides a nucleotide sequence encoding said isolated amino acid sequence.

The present invention also provides a nucleic acid construct, which contains the nucleotide sequence encoding the isolated amino acid sequence of the present invention, and is used for expressing the isolated amino acid sequence.

The present invention also provides a pharmaceutical composition, which contains the isolated amino acid sequence of the present invention and a pharmaceutically acceptable carrier.

The present invention also provides the use of the isolated amino acid sequence in the preparation of medicines for treating or preventing diseases mediated by PI3K/Akt and/or MAPK/Erk signaling pathways.

In a specific embodiment, the use is for the preparation of a medicament for treating or preventing cancer or cancer metastasis mediated by PI3K/Akt and/or MAPK/Erk signaling pathways.

In a specific embodiment, said cancer includes gastric cancer, breast cancer, colon cancer, skin cancer and kidney cancer.

In a specific embodiment, said cancer is melanoma.

Description of drawings

Figure 1 shows that the polypeptide of PAQR3 can inhibit PI3K/Akt and MAPK/ErK signaling pathways.

Figure 2 shows that fragments of PAQR3 can inhibit the migration ability of AGS cells.

Figure 3 shows that MTT and clone formation experiments prove that PAQR3 fragments can inhibit the growth of gastric cancer cells. In the figure, day0, day1, day2, day3, and day4 refer to days 0, 1, 2, 3, and 4, respectively.

Figure 4 shows that fragments of PAQR3 are able to inhibit subcutaneous tumor growth of AGS cells. In the figure, day1, day3, day4, day7, day9, and day11 refer to days 1, 3, 5, 7, 9, and 11, respectively.

Figure 5 shows that fragments of PAQR3 can inhibit the growth (A, B and C), migration (D and E) of melanoma cells A375 and the phosphorylation levels of Akt and Erk in A375 cells (F). In the figure, day0, day1, day2, day3, and day4 refer to days 0, 1, 2, 3, and 4, respectively; "control peptide-1", "control peptide-4", "control peptide-20" and "PAQR3 peptide -1", "PAQR3 Peptide-4" and "PAQR3 Peptide-20" to the control peptide and PAQR3 peptide at concentrations of 1, 4 and 20 μg/ml, respectively.

detailed description

The present invention relates to an isolated amino acid sequence, which is a fragment of SEQ ID NO: 1, which is located between the 1st and 70th positions of SEQ ID NO: 1, and has at least 40 amino acid residues in length.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state. "Isolated amino acid sequence" means that the amino acid sequence is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.

The term "fragment" refers to a polypeptide that consists of only a portion of the complete full-length polypeptide sequence and structure. The present invention relates to fragments of PAQR3. The amino acid sequence of PAQR3 is shown in SEQ ID NO:1. In particular, the present invention relates to an N-terminal fragment of PAQR3, specifically a fragment between amino acid residue 1 and amino acid 70 of SEQ ID NO: 1, preferably at least 40 amino acid residues in length, for example at least 45 amino acids residues, at least 48 amino acid residues, at least 50 amino acid residues, at least 53 amino acid residues, etc.

Preferably, the fragment at least contains the amino acid residues 6 to 55 of SEQ ID NO:1. Thus, the fragment may start at amino acid residue 1, 2, 3, 4 or 5 of SEQ ID NO: 1 and end at amino acid residue 55, 56, 57, 58, 59, 60 of SEQ ID NO: 1 , 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acid residues, ie 1-55, 1-56, 2-55, 2-56, and the like. Preferably, the fragment consists of amino acid residues 6-55 of SEQ ID NO:1.

It should be understood that in gene cloning operations, it is often necessary to design appropriate restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, which does not affect the activity of the target sequence. Another example is to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminal, C-terminal or the recombinant protein. Other suitable regions within the protein include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. The amino-terminal or carboxy-terminal of the amino acid sequence of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable label can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE and Ty1. These tags can be used to purify proteins. The table below lists some of these tags and their sequences.

The amino acid sequence of the present invention may also contain a peptide segment promoting membrane penetration at the N-terminus of the fragment. Such peptides are well known in the art, see, for example, Siegmund Reissmann, Cell penetration: scope and limitations by the application of cell-penetrating peptides, J. Pept. Sci. 2014; 20:760-784. This article incorporates its entire content into this article by reference. In particular, the membrane-penetrating-promoting peptides suitable for the present invention include the sequences numbered 1-92 listed in Table 1 of the above literature. This article does not list them all here. Peptides that can also be used in the present invention to promote membrane penetration include RKKRRQRRR.

Accordingly, the isolated amino acid sequence of the present invention may consist of said fragment of SEQ ID NO: 1, or may consist of said fragment of SEQ ID NO: 1 and a suitable protein tag, or may consist of said fragment of SEQ ID NO: 1 in combination with The sequence that facilitates membrane penetration consists of, or may consist of, the fragment of SEQ ID NO: 1, a protein tag, and a sequence that facilitates membrane penetration.

The amino acid sequences of the present invention may be chemically synthesized products, or recombinant polypeptides produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be the same as the cDNA sequence shown in SEQ ID NO:2 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes the amino acid sequence of the present invention but differs from the sequence shown in SEQ ID NO:2.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The nucleotide sequence of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the amino acid sequence of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology. Preferably, the vector of the invention is an expression vector.

The polynucleotide sequences of the present invention can be used to express or produce the amino acid sequences of the present invention by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide of the present invention or its degenerate variant, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

The polynucleotide sequences of the present invention can be inserted into recombinant expression vectors. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Methods well known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said nucleic acid sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes black Tumor cells, animal cells, etc.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

As mentioned above, previous studies have demonstrated that PAQR3 can bind to the catalytic subunit p110 of PI3K and Raf kinase, thereby inhibiting PI3K/Akt and MAPK/Erk signaling pathways. However, the present invention finds that the important functional region of PAQR3 to block the binding of p110α to p85 and the binding of Raf to MEK is at the N-terminal of PAQR3 protein, which is the fragment of SEQ ID NO: 1 described in the present invention.

Therefore, the amino acid sequence of the present invention can be used to treat or prevent various diseases known to be treatable or preventable by PAQR3, especially various diseases mediated by PI3K/Akt and/or MAPK/Erk signaling pathways. More specifically, the amino acid sequence of the present invention can be used to treat or prevent various diseases mediated by the combination of the catalytic subunit p110α of PI3K and p85 and/or the combination of Raf (especially Raf-1) and MEK.

As mentioned previously, the PI3K/Akt signaling pathway is known to be an important regulator of cell proliferation and survival. Mutations in the PIK3CA gene that result in increased PIK3 kinase activity have been reported in many human cancers, including those of the colon, breast, brain, liver, stomach, and lung [Pal I, (2012) Mandal M. PI3K and Akt as molecular targets for cancer therapy: current clinical outcomes, Acta Pharmacol Sin 33(12):1441-58]. The extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) signaling pathway is a classic signal transduction pathway, which has multiple functions such as regulating cell proliferation, differentiation, apoptosis, etc. It is closely related to prognosis and is of great significance in clinical diagnosis and treatment, especially closely related to the onset and evolution of digestive system tumors [Shen Yan et al., ERL/MAPK signal transduction pathway and digestive system tumors, "Gastroenterology", 2011, Volume 16, Issue 2].

Therefore, the amino acid sequence of the present invention is especially suitable for treating and/or preventing cancers of the colon, breast, brain, liver, skin, stomach and lung, and the metastasis of cancers occurring in these parts to other tissues of the human body. More specifically, the diseases applicable to the treatment of the amino acid sequence of the present invention include but not limited to gastric cancer, breast cancer, colon cancer, skin cancer and kidney cancer. In a specific embodiment, said cancer is gastric cancer and melanoma.

The present invention therefore includes the use of the amino acid sequence of the present invention in the preparation of a medicament for treating or preventing diseases mediated by PI3K/Akt and/or MAPK/Erk signaling pathways. Furthermore, the present invention includes that the amino acid sequence is used in the preparation of a drug for treating or preventing diseases mediated by the combination of the catalytic subunit p110α of PI3K and p85 and/or the combination of Raf (especially Raf-1) and MEK use in .

More specifically, the present invention includes the use of the amino acid sequence in the preparation of medicines for treating or preventing cancer and cancer metastasis mediated by PI3K/Akt and/or MAPK/Erk signaling pathways.

The present invention also provides a pharmaceutical composition, which contains the amino acid sequence of the present invention and a pharmaceutically acceptable carrier.

The therapeutically or prophylactically effective amount of the amino acid sequence of the present invention may be contained in the pharmaceutical composition. "Effective amount" means an amount of an ingredient sufficient to produce the desired response. The specific effective amount depends on factors such as the particular condition to be treated, the physical condition of the patient (such as the patient's weight, age, or sex), the duration of treatment, co-administered therapies (if any), and the specific drug used. formula. "Effective amount" also means that under this dosage, the toxicity or negative effect of the amino acid sequence of the present invention is less than the positive therapeutic effect brought by it.

Pharmaceutically acceptable carriers are generally safe and non-toxic, and can broadly include any known substances used in the pharmaceutical industry to prepare pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, Glidants, stabilizers, colorants, wetting agents, disintegrants, etc. The mode of administration of the pharmaceutical composition should be considered primarily in the selection of suitable excipients for the delivery of synthetic peptides, which are well known to those skilled in the art.

The content of the amino acid sequence in the pharmaceutical composition of the present invention is about 1-1000 μM; preferably about 10-500 μM; more preferably about 25-250 μM. For example, the concentration of the synthetic peptide can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 ,65,70,75,80,85,90,95,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950 or 1,000 μM.

The above-mentioned pharmaceutical compositions can be prepared according to known pharmaceutical procedures, for example, "Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences (17th Edition), edited by Alfonoso R. Gennaro, Mack Publishing Company, Easton , Pennsylvania (1985)) has a detailed record in the book.

The pharmaceutical composition of the present invention can be in various suitable dosage forms, including but not limited to tablets, capsules, injections and the like.

The present invention also provides a disease treatment or prevention method, comprising administering the amino acid sequence of the present invention or a pharmaceutical composition containing the amino acid sequence to an individual in need of the treatment or prevention. The disease is a disease mediated by PI3K/Akt and/or MAPK/Erk signaling pathway; further, the disease is the combination of the catalytic subunit p110α of PI3K and p85 and/or the combination of Raf (especially Raf-1) and Diseases mediated by MEK binding. The individual is typically a mammal, more specifically a human. The administration methods can be various administration methods known in the art, including but not limited to oral administration, injection and the like.

The present invention will be described below in the form of specific examples. The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, known to those skilled in the art. These techniques are fully explained in the literature. See, eg, Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition, 1989 et al.

Materials and Methods:

1.1 Materials:

AGS gastric cancer cells, MKN45 gastric cancer cells, and PLD1 gastric cancer cells were preserved in our laboratory; F12K, DMEM medium, and fetal bovine serum were purchased from Invitrogen Biological Company, MTT was purchased from Sigma Company, and Transwell chamber was purchased from Corning Biotechnology Company. Rats were purchased from Slack Experimental Animal Co., Ltd.; antibodies used in this experiment: p-Akt (S473), p-Erk, Akt, Erk were purchased from Cell Signaling Technology Biological Company, ki67 was purchased from BD Biological Company, and β-actin was purchased from Santa Cruz Biotechnology.

1.2 Method

1.2.1 Peptide sequence design: According to our previous research on the binding of PAQR3 to P110 and Raf-1, we designed the polypeptide of PAQR3. The polypeptide sequences of the control polypeptide and PAQR3 protein are as follows:

Control peptide: RKKRRQRRR (SEQ ID NO: 4);

Experimental peptide:

RKKRRQRRRLKSAHYIELGSYQYWPVLVPRGIRLYTYEQIPGSLKDNPYITDGYRAYLP (aka PAQR3 peptide, SEQ ID NO: 3).

The above sequence was commissioned to be synthesized by a commercial company.

1.2.2 Western blot experiment: Extract the protein of the corresponding cells, carry out SDS-PAGE, transfer to PVDF membrane and block with 5% milk powder for 2 hours, add diluted Erk1/2, p-Erk1/2, p-Akt, Akt antibody (diluted at 1:1000), overnight at 4°C, after washing with TBST, add secondary antibody HRP-labeled goat anti-mouse IgG (diluted at 1:5000), incubate at room temperature for 1 h, wash with TBST and develop with β-actin as an internal reference.

1.2.3 Detection of cell viability by MTT method: Take 2000-3000 cells/well of the above gastric cancer cell lines, inoculate them in 96-well plates, add control peptide and PAQR3 peptide at concentrations of 4 μg/mL, 20 μg/mL, daily Replace with fresh medium containing peptides. MTT was added on days 0, 1, 2, 3, and 4 to detect cell viability.

1.2.4 Colony formation experiment: 400 AGS cells, MKN45 cells, and PLD1 cells were inoculated in 6-well plates, and medium containing control polypeptide and PAQR3 peptide were added, and the medium was changed once a day. After 10 days, the medium in the well plate was discarded, fixed with 4% paraformaldehyde for 10 minutes, stained with crystal violet staining solution, photographed and counted the number of clones.

1.2.5 Migration test: After washing 40,000 AGS cells three times with serum-free medium, they were inoculated in Transwell chambers, medium containing 10% serum was added to the lower chamber, and corresponding concentrations of control polypeptide and PAQR3 peptide, take out the small chamber after 24 hours, fix it with 4% paraformaldehyde for 10 minutes, and then stain it with crystal violet staining solution, and gently wipe the cells in the upper chamber with a cotton swab, observe and take pictures of the cells under a microscope, and count the migration of cells Condition.

1.2.6 Nude mouse subcutaneous tumor and immunohistochemical experiment: 5×10 ⁶ cells were planted subcutaneously in nude mice. After the subcutaneous tumor grew to 100 mm ³ , the control peptide and PAQR3 peptide were injected into the tumor every day, 100 μg per day, Twelve days after the total injection, subcutaneous tumors were taken, embedded in paraffin and sectioned for immunohistochemical detection. Ki67 was used to mark the corresponding proliferating cells and photographed for analysis.

result:

1. PAQR3 peptide can inhibit PI3K/Akt and MAPK/Raf signaling pathways

First, in order to verify that the peptide of PAQR3 can replace the protein of PAQR3 and inhibit the PI3K/Akt and MAPK/Raf signaling pathways, we used the control peptide and PAQR3 peptide to act on AGS cells for 12 hours, then extracted the protein of each group of cells, and performed Western Blot detection . The results showed that PAQR3 peptide can effectively inhibit the levels of p-Akt(S473) and p-Erk in the three cell lines, which indirectly proved that PAQR3 peptide can replace PAQR3 protein to inhibit PI3K/Akt and MAPK/Raf signaling pathways in gastric cancer cells (figure 1).

2. PAQR3 peptide can inhibit the growth of gastric cancer cells

In order to verify whether the polypeptide of PAQR3 can replace the function of PAQR3 and inhibit the growth of cells, we verified it by MTT and clone formation experiments. The results showed that compared with the effect of the control polypeptide, the PAQR3 peptide could effectively inhibit the growth of AGS, MKN45 and PLD1 cells ( FIG. 3 ). Figures 3A-3C show the results of colony formation experiments; Figures 3D-3F show the results of MTT experiments.

3.PAQR3 peptide inhibits the migration of AGS cells

Patients with stomach cancer often have metastases, a severe exacerbation. In order to verify whether the PAQR3 peptide can inhibit the migration of gastric cancer cells, we confirmed it by Transwell experiments, and the results showed that the PAQR3 peptide can effectively inhibit the migration rate of AGS cells (Figure 2).

4. PAQR3 can inhibit the growth of subcutaneous tumor in nude mice

In order to further confirm that PAQR3 can inhibit the growth of gastric cancer cells, we first established a subcutaneous tumor model of AGS cells in nude mice. After the tumor grew to 100 mm ³ , they were divided into two groups, and 100 μg of the corresponding control peptide and PAQR3 peptide were injected into the tumor every day. , the volume of subcutaneous tumors in nude mice was measured every other day. After 12 days, the tumors were removed for measurement and photographed (Fig. 4A). The tumors of each group were embedded in paraffin, and sections were used for immunohistochemical detection, and Ki67 protein, a cell growth indicator, was labeled. The results showed that the growth of subcutaneous tumors in nude mice was effectively inhibited after treatment with PAQR3 peptide (Fig. 4B-4D).

In addition, the applicant's experiments also found that the PAQR3 peptide can effectively inhibit the growth and metastasis of human malignant melanoma A375 cells by inhibiting PI3K/Akt and MAPK/Erk signaling pathways. Specifically, the MTT method (concentrations of the control peptide and PAQR3 peptide were 1 μg/mL, 4 μg/mL, and 20 μg/mL, respectively) and the colony formation method (concentrations of the control peptide and PAQR3 peptide were 4 μg/mL and 20 μg/mL) Whether the PAQR3 peptide can inhibit the growth of A375 cells is tested, and whether the PAQR3 peptide can inhibit the migration of A375 cells is tested by scratch test and transwell chamber method (concentration of 4 μg/mL of control peptide and PAQR3 peptide is 20 μg/mL) ability. Western blot was also used to detect the levels of p-Akt and p-Erk in A375 cells under the treatment conditions of control peptide and PAQR3 peptide (control peptide: 4 μg/mL; PAQR3 peptide: 20 μg/mL).

The results are shown in Figures 5A-5F. Among them, Figures 5A-5C demonstrate that the PAQR3 peptide can inhibit the growth of A375 cells. Figures 5D and 5E demonstrate that the PAQR3 peptide has an inhibitory effect on the migration ability of A375 cells. Figure 5F demonstrates that the PAQR3 can inhibit the phosphorylation levels of Akt and Erk in A375 cells.

Claims (10)

1. An isolated amino acid sequence, characterized in that the isolated amino acid sequence contains a fragment of SEQ ID NO: 1, wherein the fragment is located between the 1st and 70th positions of SEQ ID NO: 1 and is at least 40 amino acids long Residues. 2. The isolated amino acid sequence of claim 1, wherein, The fragment is at least 45 amino acid residues in length; The fragment contains at least amino acid residues from the 6th to the 55th of SEQ ID NO:1; The amino acid sequence also contains a protein tag; and/or The amino acid sequence also contains a peptide that promotes membrane penetration. 3. The isolated amino acid sequence of claim 1 or 2, wherein The fragment consists of amino acid residues from the 6th to the 55th of SEQ ID NO:1; The isolated amino acid sequence consists of amino acid residues 6 to 55 of SEQ ID NO: 1; and/or The isolated amino acid sequence consists of the fragment of SEQ ID NO: 1 and a peptide that promotes membrane penetration. 4. The isolated amino acid sequence as claimed in claim 2 or 3, wherein the peptide that promotes membrane penetration is selected from: RQIKIWFQNRRMKWKK、YGRKKRRQRRR、KQAIPVAK-酰胺、RRRRNRTRRNRRRVR-酰胺、寡精氨酸(R ₉ －R ₁₂ )、KLTRAQRRAAARKNKRNTRGC、ALWKTLLKKVLKAPKKKRKVC、RKKRRQRRR、DAATATRGRSAASRPTERPRAPARSASRPRRPVE、GWTLNSAGYLLGKINLKALAALAKKIL-酰胺、AGYLLGKINLKALAALAKKIL-酰胺和YTAIAWVKAFIRKLRK-酰胺。 5. The isolated amino acid sequence of claim 1, wherein the isolated amino acid sequence is as shown in SEQ ID NO:3. 6. A nucleotide sequence encoding the isolated amino acid sequence of any one of claims 1-5. 7. A nucleic acid construct, characterized in that the nucleic acid construct comprises the nucleotide sequence according to claim 6. 8. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the isolated amino acid sequence according to any one of claims 1-5 and a pharmaceutically acceptable carrier. 9. The isolated amino acid sequence according to any one of claims 1-5 or the pharmaceutical composition according to claim 8 is used for the treatment or prevention of diseases mediated by PI3K/Akt and/or MAPK/Erk signaling pathways Uses in medicine. 10. The use according to claim 9, wherein the disease is cancer or cancer metastasis mediated by PI3K/Akt and/or MAPK/Erk signaling pathways, preferably, the cancer is selected from gastric cancer, breast cancer , colon, skin and kidney cancers.