CN115850388B - lncRNA encoded anticancer peptide AC115619-22AA and application thereof - Google Patents

Abstract

The application discloses an anticancer peptide AC115619-22AA coded by lncRNA and application thereof. The application belongs to the technical field of polypeptide medicaments in biochemistry, and particularly relates to an anticancer peptide AC115619-22AA coded by lncRNA and application thereof. The amino acid sequence of the AC115619-22AA polypeptide is SEQ ID No.2 in a sequence table, and the peptide acts on liver cancer cells after being dissolved in ultrapure water, can effectively inhibit the characteristics of tumor stem cells, inhibit proliferation and migration of the liver cancer cells and induce apoptosis of the liver cancer cells, can be used for preparing medicines with tumor cell inhibition activity or antitumor activity, and can be used as candidate components or material basis of medicines for treating liver cancer.

Description

lncRNA encoded anticancer peptide AC115619-22AA and application thereof

Technical Field

The application belongs to the technical field of polypeptide medicaments in biochemistry, and particularly relates to an anticancer peptide AC115619-22AA coded by lncRNA and application thereof.

Background

The overall effect of patients with middle and late stage liver cancer is poor, the death rate is up to 80%, the median survival time is less than 1 year, and the survival rate is less than 20% in 5 years. Conventional liver cancer therapeutic drugs are mostly chemotherapy drugs and small molecule targeting drugs, and the drugs have large toxic and side effects and are easy to produce drug resistance. While drugs such as immune checkpoint inhibitors have definite effects, but are limited by the immune microenvironment of patients, and the response rate is not more than 30%. Therefore, development of a novel drug for treating liver cancer is necessary.

Long non-coding RNA (lncRNA) is a protein-free coding RNA of 200 or more bases. While lncRNA has not been able to encode in traditional theory, in recent years, there has been growing evidence that lncRNA is not completely free of amino acid encoding capability, and although it cannot encode proteins of macromolecules, many lncRNA can encode small polypeptide fragments of less than 100 amino acids through a small open reading frame (small open read frame, sORF), and polypeptides encoded by these lncRNA also play an important role in the biological processes of tumors. For example, some lncRNA encoded polypeptides may inhibit tumor development. The polypeptide can be used as anticancer peptide medicine, and has the effect of inhibiting tumor by supplementing the polypeptide. Therefore, the identification and utilization of the anticancer polypeptides encoded by the lncRNA have great clinical significance for preventing and treating tumors.

Disclosure of Invention

The technical problem to be solved by the present application is how to obtain lncRNA which can encode polypeptides inhibiting tumor growth.

In order to solve the problems, the application provides a polypeptide AC115619-22AA coded by lncRNA-AC115619 and application thereof in preparing medicaments for treating liver cancer.

The present application first provides a polypeptide.

The polypeptide provided by the application is a polypeptide with an amino acid sequence of SEQ ID No.2 in a sequence table.

The application also provides a medicinal salt of the polypeptide.

Pharmaceutically acceptable salts of the polypeptides of the application, including acetate (acetate), lactobionate (lactobionate), benzenesulfonate (benzosulfonate), laurate (laurate), benzoate (benzoate), malate (malate), bicarbonate (bicarbonate), maleate (maleate), bisulfate (disulfate), mandelate (mangetate), bitartrate, methanesulfonate (mesylate), borate (borate), bromomethane (methyl bromide), bromide (bromide), methyl nitrate (methyl nitrate), calcium edetate (calcium acetate), methylsulfate (methyl sulfate), camphorsulfonic acid (camsylate), muciate (mulate), carbonates (carbonate), naphthalenesulfonates (napsylate), chlorides (chloride), nitrates (nitate), clavulanates (clavulanate), N-methylglucamine (N-methylglucamine), citrates (citrate), ammonium salts (amminium salt), dihydrochloride (dihydrochloride), oleates (oleates), ethylenediamine tetraacetate (edetate), oxalates (oxalate), ethanedisulfonates (edisylate), pamates (pamoate), propionate lauryl sulfate (estolate), palmates (palmate), ethanesulfonates (esylate), pantothenates (panthenoxylates), fumarates (fumarates), phosphates/diphosphates (phosphate/diphosphate), glucoheptonate, polygalacturonate, gluconate, salicylate, glutamate, stearate, p-hydroxyacetaminophenylarsonic acid, sulfate, hydroxybenzoate, basic acetate, sea-tangle, succinate, hydrobromide, tanninate, hydrochloride, tartrate, hydroxynaphthoate, 8-chlorotheophyllinate, iodide, tosylate, triethyliodide, lactate, valerate, etc. Depending on the application, pharmaceutically acceptable salts may be formed with cations such as sodium (sodium), potassium (potassium), aluminum (aluminum), calcium (calcium), lithium (lithium), manganese (magnesium) and zinc (zinc), bismuth (bismputh), etc., and bases such as ammonia, ethylenediamine (ethylenediamine), N-methyl-glutamine (N-methyl-glutamine), lysine (lysine), arginine (arginine), ornithine (ornitine), choline (choline), N '-dibenzylethylenediamine (N, N' -dibenzylethylenediamine), chloroprocaine (chlorprocaine), diethanolamine (dimethanol amine), procaine (procaine), diethylamine (diethylamine), piperazine (piperazine), trimethylol aminomethane (tris (hydroxymethyl)) and tetramethylammonium hydroxide (tetramethylammonium hydroxide), etc. These salts can be prepared by standard methods, for example by reaction of the free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, an acidic salt such as hydrochloride (hydrobromide), hydrobromide (hydrobromide), acetate (acetate), pamoate (pamoate) and the like can be used as the dosage form; pharmaceutically acceptable esters such as acetate, maleate, chloromethyl pivaloyloxy, etc., and esters known in the literature for improving solubility and hydrolyzability in the presence of an acidic group such as-COOH or an alcohol group, can be used as sustained release and prodrug formulations.

The application also provides a medicament with tumor cell activity inhibition or anti-tumor activity, which comprises the polypeptide or the pharmaceutically acceptable salt.

In practice, the polypeptide of the present application or a pharmaceutically acceptable salt thereof may be administered to a patient as a medicament directly or after mixing with a suitable carrier or excipient. The carrier materials herein include, but are not limited to, water soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl ethyl cellulose, etc.). Among them, preferred is a water-soluble carrier material. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injection and the like. The suppository can be pessary, vaginal ring, ointment, cream or gel suitable for vaginal application. Can be common preparation, slow release preparation, controlled release preparation and various microparticle administration systems. For the purpose of shaping the unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets. For the purpose of formulating the unit dosage form into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. For preparing a unit dosage form into a suppository, various carriers well known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. For preparing unit dosage forms into injectable preparations such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired.

The preparation can be administrated by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; administration via the luminal tract, such as rectally, vaginally, and sublingually; respiratory tract administration, such as via the nasal cavity; mucosal administration. The above route of administration is preferably injection.

In the above medicine, the tumor is liver cancer.

Among the above drugs, the drug has at least one of the following properties:

a1, preparing a product for inhibiting the activity of tumor cells;

a2, the application in preparing products with anti-tumor activity.

The application also provides application of the polypeptide in preparing a product with tumor cell inhibition activity or anti-tumor activity.

The use of the above-mentioned pharmaceutically acceptable salts for the preparation of products having tumor cell inhibiting or antitumor activity is also within the scope of the present application.

In the above application, the tumor cell is a liver cancer cell.

The application also provides a biological material, which is any one of the following:

c1 A nucleic acid molecule encoding the polypeptide of claim 1;

c2 An expression cassette comprising C1) said nucleic acid molecule;

c3 A recombinant vector comprising the nucleic acid molecule of C1) or a recombinant vector comprising the expression cassette of C2).

The application provides a polypeptide AC115619-22AA coded by lncRNA-AC115619, and researches the application of the polypeptide AC115619-22AA in preparing a medicine for treating liver cancer. The polypeptide acts on liver cancer cells after being dissolved in ultrapure water, can effectively inhibit the characteristics of tumor stem cells, inhibit proliferation and migration of the liver cancer cells, and induce apoptosis of the liver cancer cells. Therefore, the polypeptide AC115619-22AA is an anticancer peptide with liver cancer inhibition effect, and can be used as a candidate component or a substance basis of a medicament for treating liver cancer.

Drawings

FIG. 1 shows the identification of synthetic AC115619-22AA anticancer peptide by high performance liquid chromatography.

FIG. 2 is a liquid chromatography-mass spectrometry method for identifying an artificially synthesized AC115619-22AA anticancer peptide.

FIG. 3 shows the immunofluorescence of the AC115619-22AA polypeptide absorbed by liver cancer cells.

FIG. 4 shows the ability of the AC115619-22AA anticancer peptide to effectively inhibit the balling of hepatoma cells.

FIG. 5 shows the inhibition of hepatocarcinoma cells by AC115619-22AA anticancer peptide at different concentrations.

FIG. 6 shows the inhibition of liver cancer cells by 400 μg/mL of AC115619-22AA anticancer peptide under different treatment time conditions.

FIG. 7 shows the clonogenic potential of AC115619-22AA anticancer peptide in inhibiting liver cancer cell effectively.

FIG. 8 shows the induction of apoptosis of liver cancer cells at 12h with different concentrations of AC115619-22AA anticancer peptide.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.

EXAMPLE 1 Synthesis of AC115619-22AA anticancer peptide

lncRNA-AC115619 (ENST 00000567376.2) is lncRNA located on Chromosome 2 of human, and the specific Chromosome coordinates are Chromosome 2:20,999,313-21,000,917, and 1605 bases in length. The lncRNA is liver-specific lncRNA, is expressed only in liver, and exhibits low expression in liver cancer cells.

By ribosome sequencing and analysis using the orfffinder database developed by the national center for biotechnology information, a region of sORF of 66 bases in length was identified at the base 471-536 of the lncRNA, which was first confirmed to encode a polypeptide. The nucleotide sequence of the sORF is ATGGCAATACGGACCCCAGATGTCTCCCTGGAGCTCTGCCAGGATCCATTCAAGGCCAAGAAAGTA (sequence 1 in the sequence table), the translated polypeptide contains 22 amino acids, and the amino acid sequence is: met-Ala-Ile-Arg-Thr-Pro-Asp-Val-Ser-Leu-Glu-Leu-Cys-Gln-Asp-Pro-Phe-Lys-Ala-Lys-Lys-Val (methionine-alanine-isoleucine-arginine-threonine-proline-aspartic acid-valine-threonine-leucine-glutamic acid-leucine-cysteine-glutamine-aspartic acid-proline-phenylalanine-lysine-alanine-lysine-valine), the amino acid sequence of which is sequence 2 in the sequence listing, the molecular weight of which is: 2488.7kDa, this peptide is referred to as AC115619-22AA polypeptide or AC115619-22AA.

The peptide was synthesized artificially using a solid-phase chemical synthesis method according to the known amino acid sequence of AC115619-22AA. The synthesized AC115619-22AA was then identified by high performance liquid chromatography (see FIG. 1 for results) and liquid chromatography-mass spectrometry (see FIG. 2 for results).

The results show that the product purity of AC115619-22AA is >95%, and the water solubility is good. Suggesting that the AC115619-22AA polypeptide is synthesized without error.

EXAMPLE 2 uptake of fluorescein isothiocyanate FITC in combination with the AC115619-22AA polypeptide by cells

In the course of polypeptide synthesis, fluorescein isothiocyanate FITC was labeled into AC115619-22AA. HUH7 hepatoma cells (purchased from the GmbH of the Living technology Co., ltd., product number: CL-0120) were grown in 6-well plates at a density of 2 ten thousand cells/well and cultured for 24 hours in a conventional manner. FITC-labeled AC115619-22AA polypeptide was then added to DMEM medium (Withanbozier Life technologies Co., ltd., cat# PM 150210B) at a concentration of 400. Mu.g/mL and incubation was continued for 4h. As shown in FIG. 3, immunofluorescence detection shows that AC115619-22AA can be absorbed into cytoplasm by liver cancer cells, which suggests that the peptide is well absorbed by cells, can penetrate cell membranes, and has medicinal value.

Example 3 study of inhibition of the Activity of the AC115619-22AA polypeptide on the characteristics of liver cancer cell tumor Stem cells

The effect of AC115619-22AA on the characteristics of liver cancer cell tumor stem cells was observed using cell balling experiments. The experiment was repeated three times, each repetition comprising the following specific steps:

HUH7 liver cancer cells (purchased from the Ghandenox life technologies Co., ltd., product number: CL-0120) were planted in ultra-low adsorption 6-well plate cell culture plates at a density of 1 ten thousand cells/well, 3mL of DMEM medium was added, and two treatment groups, namely a polypeptide treatment group and a control group, were experimentally set. The polypeptide treatment group was added with an aqueous solution of AC115619-22AA to a final concentration of 400. Mu.g/mL, and the control group was added with an equal amount of aqueous solution (0. Mu.g/mL AC115619-22 AA). 5 wells per treatment group, placed at 37℃in 5% CO ₂ Culturing in a constant temperature incubator for 1 week, and under a mirrorAnd observing the balling condition.

Experimental results show that the cell can effectively inhibit the balling capacity of HUH7 cells by using 400 mug/mL AC115619-22AA to treat the cells, the balling capacity of the cells is weakened, the balling size is reduced (figure 4), and the AC115619-22AA is suggested to inhibit the stem cell characteristics of liver cancer cells.

EXAMPLE 4 inhibition of proliferation and Induction of apoptosis by AC115619-22AA polypeptide

1. Concentration study for inhibiting liver cancer cells by using AC115619-22AA polypeptide

The inhibition of hepatoma cell proliferation by the AC115619-22AA polypeptide was analyzed using a CCK-8 kit (purchased from Shanghai, assist Biotech Co., ltd., cat# 40203ES 60). The experiment was repeated three times, each repetition comprising the following specific steps:

1) Preparing an aqueous solution of AC115619-22 AA: an aqueous solution of AC115619-22AA (concentration: 8.0 mg/mL) was prepared using sterile, enzyme-free water as a solvent and AC115619-22AA as a solute.

2) HUH7 hepatoma cells were seeded at a density of 5000 cells/well in 96-well cell culture plates with a final liquid volume per well of 200 μl DMEM medium, and after cell attachment, 6 treatment groups of 5 wells per treatment group were experimentally set:

(1) 200. Mu.L of DMEM medium was added to a 0. Mu.g/mL AC115619-22AA treatment group and the mixture was placed at 37℃in 5% CO ₂ Culturing for 48 hours in a constant temperature incubator;

(2) adding 1.25 mu L of AC115619-22AA aqueous solution and 198.75 mu L of DMEM medium into 50 mu g/mL of AC115619-22AA treatment group, placing the mixture at 37 ℃ and 5% CO to make the content of AC115619-22AA in the culture system be 50 mu g/mL ₂ Culturing for 48 hours in a constant temperature incubator;

(3) 100 mu g/mL of AC115619-22AA treatment group is added with 2.5 mu L of AC115619-22AA aqueous solution and 197.5 mu L of DMEM culture medium, so that the content of the AC115619-22AA in the culture system is 100 mu g/mL, and the culture system is placed at 37 ℃ and 5% CO ₂ Culturing for 48 hours in a constant temperature incubator;

(4) 200 mu g/mL of AC115619-22AA treatment group is added with 5.0 mu L of AC115619-22AA aqueous solution and 195.0 mu L of DMEM culture medium, so that the content of the AC115619-22AA in the culture system is 200 mu g/mL, and the culture system is placed at 37 ℃ and 5% CO ₂ Culturing for 48 hours in a constant temperature incubator;

(5) adding 10. Mu.L of AC115619-22AA aqueous solution +190.0. Mu.L of LDMEM culture medium into 400. Mu.g/mL of AC115619-22AA treatment group to make the content of AC115619-22AA in the culture system 400. Mu.g/mL, placing at 37 deg.C and 5% CO ₂ Is cultured in a constant temperature incubator for 48 hours.

3) The CCK-8 reagent at 1:100 was used to detect changes in the activity of cells in each treatment group, and absorbance at a wavelength of 450nm was measured using a microplate reader to reflect the cell activity. Cell inhibition = 1- (detection time point absorbance/0 h time point absorbance) ×100%.

2. Treatment time study of inhibiting liver cancer cells by AC115619-22AA polypeptide

The inhibition of hepatoma cell proliferation by the AC115619-22AA polypeptide was analyzed using a CCK-8 kit (purchased from Shanghai, assist Biotech Co., ltd., cat# 40203ES 60). The experiment was repeated three times, each repetition comprising the following specific steps:

1) Preparing an aqueous solution of AC115619-22 AA: an aqueous solution of AC115619-22AA (concentration: 8.0 mg/mL) was prepared using sterile, enzyme-free water as a solvent and AC115619-22AA as a solute.

2) HUH7 hepatoma cells were seeded at a density of 5000 cells/well in 96-well cell culture plates with a final liquid content of 200 μl DMEM medium per well, and after cell attachment, the experiment was performed with 4 treatment groups:

(1) the 0 hour treatment group was added with 10. Mu.L of AC115619-22AA aqueous solution and 190.0. Mu.L of DMEM medium, and the mixture was placed at 37℃with 5% CO ₂ Culturing in a constant temperature incubator for 0 hour to detect;

(2) adding 10 μl of AC115619-22AA aqueous solution and 190.0 μl of DMEM medium into 12 hr treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL, placing at 37deg.C and 5% CO ₂ Is cultured in a constant temperature incubator for 12 hours and then is detected;

(3) adding 10 μl of AC115619-22AA aqueous solution and 190.0 μl of DMEM medium into 24 hr treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL, placing at 37deg.C and 5% CO ₂ Is cultured in a constant temperature incubator for 24 hours and then is detected;

(4) the 48 hour treatment group was added with 10. Mu.LAC115619-22AA aqueous solution +190.0. Mu.L DMEM medium, the content of AC115619-22AA in the culture system is 400. Mu.g/mL, and the culture system is placed at 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator for 48 hours and then is detected;

(5) adding 10 μl of AC115619-22AA aqueous solution and 190.0 μl of DMEM medium into 72 hr treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL, placing at 37deg.C and 5% CO ₂ Is cultured in a constant temperature incubator for 72 hours and then is detected;

each treatment group had 5 wells.

3) The CCK-8 reagent at 1:100 detects changes in the activity of cells in each treatment group, and absorbance at a wavelength of 450nm was detected using a microplate reader to reflect the cell activity, and the data were normalized at 0 concentration or at 0 time point. Cell viability= (detection time point absorbance/0 h time point absorbance) ×100%.

As shown in FIG. 5 and FIG. 6, the AC115619-22AA with the concentration of 100 mug/mL can show obvious inhibition effect on HUH7 liver cancer cells, and the survival rate on HUH7 liver cancer cells is gradually reduced along with the increase of the concentration of the AC115619-22AA polypeptide; HUH7 hepatoma cells were treated with 400. Mu.g/mL of AC115619-22AA polypeptide, which showed inhibitory effect on HUH7 hepatoma cells at 24h. Thus, the AC115619-22AA polypeptide has anticancer activity.

3. Clone formation method for analyzing inhibition effect of AC115619-22AA polypeptide on liver cancer cell proliferation

The experiment was repeated three times, each repetition comprising the following specific steps:

1) Preparing an aqueous solution of AC115619-22 AA: an aqueous solution of AC115619-22AA (concentration: 8.0 mg/mL) was prepared using sterile, enzyme-free water as a solvent and AC115619-22AA as a solute.

2) HUH7 hepatoma cells (purchased from the wumprosate life technologies company, inc., cat No.: CL-0120) were planted in 6-well plate cell culture plates at a density of 1000 cells/well, DMEM medium was added in 3mL, and after cell attachment, the experiment was performed in 3 treatment groups:

(1) a0. Mu.g/mL AC115619-22AA treatment group was added to 2000. Mu.LDMEM medium and placed at 37℃in 5% CO ₂ Culturing in a constant temperature incubator for 1 week, and changing the culture medium with DMEM every day;

(2) adding 12.5 mu L of AC115619-22AA aqueous solution and 1975.5 mu L of DMEM medium into 50 mu g/mL of AC115619-22AA treatment group, placing the mixture at 37 ℃ and 5% CO to ensure that the content of the AC115619-22AA in the culture system is 50 mu g/mL ₂ Culturing in a constant temperature incubator for 1 week, and changing the culture medium with 50 mug/mL of AC115619-22AA DMEM every day;

(3) adding 100. Mu.L of AC115619-22AA aqueous solution and 1900.0. Mu.L of DMEM medium into 400. Mu.g/mL of AC115619-22AA treatment group to make the content of AC115619-22AA in the culture system 400. Mu.g/mL, placing at 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator for 1 week, and replaced with 400. Mu.g/mL of AC115619-22AA DMEM medium every day. Clone formation was observed using crystal violet staining.

The 0. Mu.g/mL AC115619-22AA treated group resulted in 125.0.+ -. 12.2 effective clones, the 50. Mu.g/mL AC115619-22AA treated group resulted in 75.6.+ -. 6.2 effective clones, and the 400. Mu.g/mL AC115619-22AA treated group resulted in 0 effective clones. Suggesting that AC115619-22AA inhibits the clonogenic formation of hepatoma cells.

Wherein, the DMEM+50 mug/mL culture medium is a liquid obtained by adding the aqueous solution of the AC115619-22AA into the DMEM culture medium, and the content of the AC115619-22AA in the DMEM+50 mug/mL culture medium is 50 mug/mL. The DMEM+400. Mu.g/mL medium is a liquid obtained by adding the aqueous solution of AC115619-22AA to the DMEM medium, and the content of AC115619-22AA in the DMEM+400. Mu.g/mL medium is 400. Mu.g/mL.

As shown in FIG. 7, the AC115619-22AA polypeptide with the concentration of 400 mug/mL effectively inhibits the clonogenic capacity of liver cancer cells, which proves that the AC115619-22AA polypeptide has anticancer activity.

4. RealTime Glo Annexin V Apoptosis Assay real-time apoptosis monitoring kit for detecting induction of apoptosis of liver cancer cells by AC115619-22AA polypeptide

The real-time apoptosis monitoring kit (Promega biotechnology Co., ltd., product number JA 1000) detects the induction of apoptosis of hepatoma cells by AC115619-22AA, and comprises the following specific steps:

1) Preparing an aqueous solution of AC115619-22 AA: an aqueous solution of AC115619-22AA (concentration: 8.0 mg/mL) was prepared using water as a solvent and AC115619-22AA as a solute.

2) HUH7 hepatoma cells were seeded at a density of 5000 cells/well in 96-well cell culture plates with a final liquid volume per well of 200 μl DMEM medium, and after cell attachment, 6 treatment groups of 5 wells per treatment group were experimentally set:

(1) 200. Mu.L of DMEM medium was added to a 0. Mu.g/mL AC115619-22AA treatment group and the mixture was placed at 37℃in 5% CO ₂ Is cultured in a constant temperature incubator for 72 hours;

(2) 50. Mu.g/mL AC115619-22AA treatment group was added with 1.25. Mu.LAC115619-22AA aqueous solution +198.75 mu L DMEM medium, so that the content of AC115619-22AA in the culture system is 50 mu g/mL, and placing the culture system at 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator for 72 hours;

(3) 100 mu g/mL of AC115619-22AA treatment group is added with 2.5 mu L of AC115619-22AA aqueous solution and 197.5 mu L of DMEM culture medium, so that the content of the AC115619-22AA in the culture system is 100 mu g/mL, and the culture system is placed at 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator for 72 hours;

(4) 200 mu g/mL of AC115619-22AA treatment group is added with 5.0 mu L of AC115619-22AA aqueous solution and 195.0 mu L of DMEM culture medium, so that the content of the AC115619-22AA in the culture system is 200 mu g/mL, and the culture system is placed at 37 ℃ and 5% CO ₂ Is cultured in a constant temperature incubator for 72 hours;

(5) adding 10. Mu.L of AC115619-22AA aqueous solution +190.0. Mu.L of LDMEM culture medium into 400. Mu.g/mL of AC115619-22AA treatment group to make the content of AC115619-22AA in the culture system 400. Mu.g/mL, placing at 37 deg.C and 5% CO ₂ Is cultured in a constant temperature incubator for 72 hours.

Apoptosis signals were detected every 12h using a bioluminescence detector (spar 10M, open sea trade limited).

As shown in FIG. 8, the treatment of 50 mug/mL of AC115619-22AA can cause apoptosis of liver cancer cells at 12h, and the apoptosis is more obvious along with the increase of the concentration of AC115619-22AA.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (8)

1. A polypeptide, characterized in that: the polypeptide is a polypeptide with an amino acid sequence of SEQ ID No.2 in a sequence table.

2. A pharmaceutically acceptable salt of the polypeptide of claim 1.

3. A medicament having an inhibitory activity against tumor cells or an antitumor activity, characterized in that: the medicament contains the polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2.

4. A medicament according to claim 3, characterized in that: the tumor is liver cancer.

5. The medicament according to claim 3 or 4, characterized in that it has at least one of the following properties:

a1, preparing a product for inhibiting the activity of tumor cells;

a2, the application in preparing products with anti-tumor activity.

6. Use of the polypeptide of claim 1 for the preparation of a product having an inhibitory activity or an anti-tumor activity against a tumor cell, said tumor cell being a liver cancer cell.

7. The use of a pharmaceutically acceptable salt according to claim 2 for the preparation of a product having an inhibitory or anti-tumor activity against a tumor cell, which is a liver cancer cell.

8. A biomaterial characterized in that the biomaterial is any one of the following:

c1 A nucleic acid molecule encoding the polypeptide of claim 1;

c2 An expression cassette comprising C1) said nucleic acid molecule;

c3 A recombinant vector comprising the nucleic acid molecule of C1) or a recombinant vector comprising the expression cassette of C2).