CN115850388A - lncRNA coded anti-cancer peptide AC115619-22AA and application - Google Patents

Abstract

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

Description

lncRNA coded anti-cancer peptide AC115619-22AA and application

Technical Field

The invention belongs to the technical field of polypeptide medicines in biochemistry, and particularly relates to an lncRNA-encoded anti-cancer peptide AC115619-22AA and application thereof.

Background

The overall effect of the patients in the middle and late stages of liver cancer is poor, the mortality rate is up to 80 percent, the median survival time is less than 1 year, and the 5-year survival rate is less than 20 percent. Most of conventional liver cancer treatment medicines are chemotherapeutic medicines and small-molecule targeted medicines, and the medicines have large toxic and side effects and are easy to generate drug resistance. While drugs such as immune checkpoint inhibitors have clear effects, but are limited by the immune microenvironment of patients, and the response rate does not exceed 30%. Therefore, there is a need to develop new drugs for treating liver cancer.

Long non-coding RNA (lncRNA) is a protein-free coding RNA composed of more than 200 bases. The conventional theory considers that lncRNA has no coding capacity, but in recent years, more and more evidences prove that lncRNA does not have completely no amino acid coding capacity, and although large molecular proteins cannot be coded, many lncRNA can code small polypeptide fragments with less than 100 amino acids through a small open reading frame (sORF), and the polypeptides coded by these lncRNA also play an important role in the biological process of tumors. For example, some lncRNA-encoded polypeptides may inhibit tumor development. The polypeptide can be used as an anticancer peptide drug, and has the effect of inhibiting tumors by supplementing the polypeptide. Therefore, the identification and the utilization of the anti-cancer polypeptides coded by the lncRNA have great clinical significance for preventing and treating tumors.

Disclosure of Invention

The technical problem to be solved by the invention is how to obtain the lncRNA which can code the polypeptide for inhibiting the tumor growth.

In order to solve the problems, the invention provides a polypeptide AC115619-22AA coded by lncRNA-AC115619, and provides application of the polypeptide AC115619-22AA in preparation of a medicine for treating liver cancer.

The invention provides a polypeptide.

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

The invention also provides a medicinal salt of the polypeptide.

Pharmaceutically acceptable salts of the polypeptides of the invention include acetate (acetate), lactobionate (lactobionate), benzenesulfonate (benzylsulfonate), laurate (laurate), benzoate (benzoate), malate (malte), bicarbonate (bicarbonate), maleate (maleate), bisulfate (bisulfate), mandelate (mandelate), bitartrate (bitartrate), methanesulfonate (mesylate), borate (borate), methyl bromide (bromide), methyl nitrate (methylnitrate), calcium edetate (caldate), methylsulfate (methylsulfate), camsylate (camphorsulfonate), mucate (mucate), carbonates (carbonates), naphthalenesulfonates (napsylates), chlorides (chlorides), nitrates (nitrates), clavulanates (clavulanates), N-methylglucamines (N-methylglucamines), citrates (citrates), ammonium salts (ammonium salts), dihydrochloride (dihydrochlorides), oleates (oleates), ethylenediaminetetraates (etates), oxalates (oxalates), pamoates (pamoates), propionates (laurates), palmitates (palmitates), ethanesulfonates (esylates), pantothenate (pantoates), phosphates/diphosphates (phosphates/diphosphates), glucoheptonate (gluceptate), polygalacturonate (polygalacturonate), glucoheptonate (gluconate), salicylate (salicylate), glutamate (glutamate), stearate (stearate), hydroxyacetaminophenylarsonate (glycollylarsanilate), sulfate (sulfate), hydroxybenzoate (hexahydrobenzoate), subacetate (subacetate), hydrabamine (hydrabamine), succinate (succinate), hydrobromide (hydrobromide), tannate (tannate), hydrochloride (hydrochloride), tartrate (tartate), hydroxynaphthoate (hydroxynaphthoate), 8-chlorothalonium salt (teoclate), iodide (iodide), tosylate (tosylate), triiodode (triiodode), lactate (vallate), valerate (valerate), and the like. Depending on the use, pharmaceutically acceptable salts may be formed from cations such as sodium (sodium), potassium (potassium), aluminum (aluminum), calcium (calcium), lithium (lithium), manganese (magnesium), and zinc (zinc), bismuth (bismuth), and the like, or bases such as ammonia, ethylenediamine (ethylenediamine), N-methyl-glutamine (N-methyl-glutamine), lysine (lysine), arginine (arginine), ornithine (ornithine), choline (choline), N '-dibenzylethylenediamine (N, N' -dibenzylethylenediamine), chloroprocaine (chloroprocaine), diethanolamine (diethanolamine), procaine (procaine), diethylamine (diethylamine), piperazine (piperazine), tris (hydroxymethyl) aminomethane (tetramethylammonium), and hydroxylamine (hydroxylamine), and the like. 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, acetate, pamoate and the like may be used as the dosage form; pharmaceutically acceptable esters such as acetate (acetate), maleate (maleate), chloromethyl (pivaloyloxymethyl) acetate, and the like, 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 invention also provides a medicament with tumor cell inhibiting activity or anti-tumor activity, which contains the polypeptide or the medicinal salt.

In practice, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof may be administered as a drug directly to a patient, or may be administered to a patient after being mixed with a suitable carrier or excipient. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. 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 injections and the like. Wherein the suppository can be vaginal suppository, vaginal ring, ointment, cream or gel suitable for vaginal application. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier 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 and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layered and multi-layered tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. For preparing the unit dosage form into preparations for injection such as solution, emulsion, lyophilized powder and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.

The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, and the like; for buccal administration, e.g., rectally, vaginally, and sublingually; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above route of administration is preferably by injection.

In the above medicine, the tumor is liver cancer.

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

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

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

The invention also provides the application of the polypeptide in preparing products with tumor cell inhibiting activity or anti-tumor activity.

The application of the medicinal salt in the preparation of products with tumor cell inhibiting activity or anti-tumor activity also belongs to the protection scope of the invention.

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

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

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

c2 An expression cassette comprising the nucleic acid molecule according to C1);

c3 A recombinant vector containing the nucleic acid molecule according to C1) or a recombinant vector containing the expression cassette according to C2).

The invention provides a polypeptide AC115619-22AA coded by lncRNA-AC115619, and researches the application of the polypeptide AC115619-22AA in preparing a medicament for treating liver cancer. The polypeptide is dissolved in ultrapure water and then acts on liver cancer cells, so that the characteristics of the tumor stem cells can be effectively inhibited, the proliferation and migration of the liver cancer cells are inhibited, and the apoptosis of the liver cancer cells is induced. Therefore, the polypeptide AC115619-22AA is an anticancer peptide with liver cancer inhibition effect, and can be used as a candidate component or a material basis of a liver cancer treatment drug.

Drawings

FIG. 1 is a high performance liquid chromatography method for identifying artificially synthesized AC115619-22AA anticancer peptides.

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

FIG. 3 shows the situation that the AC115619-22AA polypeptide is absorbed by hepatoma carcinoma cells through immunofluorescence detection.

FIG. 4 shows that AC115619-22AA anticancer peptide can effectively inhibit the balling-up ability of hepatoma carcinoma cells.

FIG. 5 shows the inhibitory effect of different concentrations of AC115619-22AA anticancer peptide on hepatoma cells.

FIG. 6 shows the inhibition of hepatoma cells by 400. Mu.g/mL of AC115619-22AA anticancer peptide under different treatment time conditions.

FIG. 7 shows that the AC115619-22AA anticancer peptide can effectively inhibit the clonogenic capacity of hepatoma carcinoma cells.

FIG. 8 shows that different concentrations of AC115619-22AA anticancer peptide induced apoptosis of hepatoma cells at 12 h.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative experiments in the following examples, unless otherwise specified, were set up in triplicate.

Example 1 Synthesis of AC115619-22AA anticancer peptides

lncRNA-AC115619 (ENST 00000567376.2) is lncRNA located on human Chromosome 2 with specific chromosomal coordinates Chromosome 2. The lncRNA is liver-specific lncRNA, is only expressed in the liver, and is low expressed in liver cancer cells.

Through ribosome sequencing and analysis by using an ORFfinder database developed by the national center for biotechnology information, an sORF region with the length of 66 bases is identified at the positions of 471-536 bases of the lncRNA, and the sORF region is proved to encode polypeptide for the first time. The nucleotide sequence of the sORF is ATGGCAATACGGACCCAGATGTCCCTGGAGCTGTCCAGGATCCATTCAAGGCCAAGAAAGTA (sequence 1 in a sequence table), the translated polypeptide contains 22 amino acids, and the amino acid sequence is as follows: 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 is sequence 2 in the sequence table, and the molecular weight is: 2488.7kDa, this peptide is referred to as AC115619-22AA polypeptide or AC115619-22AA.

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

The results show that the product purity of AC115619-22AA is >95%, and the water solubility is good. The synthesis of the AC115619-22AA polypeptide is not correct.

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

In the polypeptide synthesis, fluorescein isothiocyanate FITC was labeled to AC115619-22AA. HUH7 hepatoma cells (purchased from GmbH, K.K.: CL-0120) were seeded at a density of 2 ten thousand per well in 6-well plates and cultured routinely for 24h. Subsequently, FITC-labeled AC115619-22AA polypeptide was added to a DMEM medium (Wuhan Protech technologies, ltd., product number: PM 150210B) at a concentration of 400. Mu.g/mL, and the culture was continued for 4 hours. As shown in FIG. 3, immunofluorescence assay found that AC115619-22AA can be absorbed into the cytoplasm by hepatoma cells, suggesting that the peptide is well absorbed by the cells, can penetrate cell membranes, and has medicinal value.

Example 3 study of inhibitory Effect of AC115619-22AA Polypeptides on characteristics of hepatoma cell tumor stem cells

The effect of AC115619-22AA on the characteristics of the liver cancer cell tumor stem cells is observed by using a cell balling experiment. The experiment was repeated three times, each time with the following specific steps:

HUH7 liver cancer cells (purchased from wuhan pusunosike life science ltd, cat #: CL-0120) was plated at a density of 1 ten thousand per well on an ultra-low adsorption 6-well plate cell culture plate, 3mL of DMEM medium was added, and two treatment groups, a polypeptide treatment group and a control group, were used for the experiment. The polypeptide-treated 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 equivalent amount of an aqueous solution (0. Mu.g/mL AC115619-22 AA). 5 wells per treatment group, at 37 ℃ and 5% CO ₂ The cells were cultured in a constant temperature incubator for 1 week, and then the beading was observed under a microscope.

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

Example 4 inhibition of AC115619-22AA Polypeptides on hepatoma cell proliferation and Induction of apoptosis

1. Study on use concentration of AC115619-22AA polypeptide for inhibiting liver cancer cells

The inhibitory effect of AC115619-22AA polypeptide on hepatoma cell proliferation was analyzed using CCK-8 kit (purchased from Shanghai assist in Sheng Biotech, inc., cat.: 40203ES 60). The experiment was repeated three times, each repetition of the specific steps as follows:

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

2) HUH7 liver cancer cells are planted in a 96-well cell culture plate at a density of 5000 cells/well, the final liquid amount of each well is 200 mu L DMEM medium, after the cells are attached to the wall, 6 treatment groups are set in an experiment, and 5 wells are arranged in each treatment group:

(1) adding 200 μ L DMEM medium to the 0 μ g/mL AC115619-22AA treated group, incubating at 37 deg.C, 5% ₂ Culturing for 48 hours in a constant-temperature incubator;

(2) the 50. Mu.g/mL AC115619-22AA treated group was added with 1.25. Mu.L of aqueous solution of AC115619-22AA + 198.75. Mu.L DMEM medium to adjust the AC115619-22AA content in the culture system to 50. Mu.g/mL, and the mixture was incubated at 37 ℃ and 5% CO ₂ Culturing for 48 hours in a constant-temperature incubator;

(3) the 100. Mu.g/mL AC115619-22AA treated group was added with 2.5. Mu.L of aqueous solution of AC115619-22AA + 197.5. Mu.L DMEM medium to make the AC115619-22AA content in the culture system 100. Mu.g/mL, and the mixture was incubated at 37 ℃ for 5% CO ₂ Culturing for 48 hours in a constant-temperature incubator;

(4) the 200. Mu.g/mL AC115619-22AA treated group was supplemented with 5.0. Mu.L of aqueous AC115619-22AA solution + 195.0. Mu.L DMEM medium to make the AC115619-22AA content in the culture system 200. Mu.g/mL, and was subjected to 5% CO at 37 ℃% ₂ Culturing for 48 hours in a constant-temperature incubator;

(5) the 400. Mu.g/mL AC115619-22AA treated group was added with 10. Mu.L of an aqueous solution of AC115619-22AA plus 190.0. Mu.L of LDMEM medium to make the AC115619-22AA content in the culture system 400. Mu.g/mL, placed at 37 ℃ and 5% CO ₂ The culture was carried out in a constant temperature incubator for 48 hours.

3) 1, CCK-8 reagent of 100 is used for detecting activity change of cells of each treatment group, and the enzyme-labeling instrument is used for detecting the absorbance at the wavelength of 450nm so as to reflect the activity of the cells. Cell inhibition =1- (absorbance at detection time/absorbance at 0h time) × 100%.

2. Study on treatment time of AC115619-22AA polypeptide for inhibiting liver cancer cells

The inhibitory effect of the AC115619-22AA polypeptide on the proliferation of hepatoma cells was analyzed using a CCK-8 kit (purchased from assist in Biotechnology Ltd, shanghai, inc., cathaya, inc.: 40203ES 60). The experiment was repeated three times, each time with the following specific steps:

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

2) HUH7 liver cancer cells are planted in a 96-well cell culture plate at the density of 5000 cells/well, the final liquid content of each well is 200 mu L DMEM medium, and after the cells adhere to the wall, 4 treatment groups are set in the experiment:

(1) 0 hour treatment group was added with 10. Mu.L of AC115619-22AA aqueous solution + 190.0. Mu.L of DMEM medium, incubated at 37 ℃ and 5% CO ₂ The detection is carried out after 0 hour of culture in a constant temperature incubator;

(2) the 12-hour treatment group was added with 10. Mu.L of aqueous solution of AC115619-22AA and 190.0. Mu.L of DMEM medium to make the AC115619-22AA content in the culture system 400. Mu.g/mL, and the mixture was allowed to stand at 37 ℃ and 5% CO ₂ The culture is carried out in a constant temperature incubator for 12 hours and then detection is carried out;

(3) adding 10 μ L of aqueous solution of AC115619-22AA and 190.0 μ L of DMEM medium to the 24-hour treatment group to make the content of AC115619-22AA in the culture system 400 μ g/mL, and removing the C at 37 deg.C and 5% CO ₂ Detecting the culture medium after culturing for 24 hours in a constant temperature incubator;

(4) the 48-hour treatment group was supplemented with 10. Mu.L of aqueous solution of AC115619-22AA in addition to 190.0. Mu.L of DMEM medium to make the AC115619-22AA content in the culture system 400. Mu.g/mL, and the mixture was incubated at 37 ℃ and 5% CO ₂ Culturing in a constant-temperature incubator for 48 hours, and detecting;

(5) adding 10 μ L of aqueous solution of AC115619-22AA and 190.0 μ L of DMEM medium to the 72-hour treatment group to make the content of AC115619-22AA in the culture system 400 μ g/mL, and removing the C at 37 deg.C and 5% CO ₂ Culturing for 72 hours in a constant-temperature incubator, and detecting;

5 wells per treatment group.

3) The CCK-8 reagent of 1. Cell viability = (absorbance at detection time point/absorbance at 0h time point) × 100%.

The results are shown in fig. 5 and fig. 6, AC115619-22AA with a concentration of 100 μ g/mL can show a more obvious inhibition effect on HUH7 liver cancer cells, and the survival rate on HUH7 liver cancer cells is gradually reduced with the increase of the concentration of AC115619-22AA polypeptide; HUH7 liver cancer cells are treated by 400 mu g/mL of AC115619-22AA polypeptide, and the inhibition effect on the HUH7 liver cancer cells is shown at 24h. Therefore, the AC115619-22AA polypeptide has anticancer activity.

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

The experiment was repeated three times, each time with the following specific steps:

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

2) HUH7 liver cancer cells (purchased from wuhan pusunosike life science ltd, cat #: CL-0120) were planted at a density of 1000/well in 6-well plate cell culture plates, 3mL of DMEM medium was added, and after cells were attached to the wall, 3 treatment groups were set:

(1) 0 μ g/mL AC115619-22AA treated group was added 2000 μ L MEM medium, incubated at 37 ℃ and 5% CO ₂ Culturing for 1 week in a constant-temperature incubator, and replacing the culture solution with DMEM every day;

(2) the 50. Mu.g/mL AC115619-22AA treated group was added with 12.5. Mu.L of an aqueous solution of AC115619-22AA plus 1975.5. Mu.L DMEM medium to make the AC115619-22AA content in the culture system 50. Mu.g/mL, incubated at 37 ℃ and 5% CO ₂ The culture is carried out in a constant temperature incubator for 1 week, and the solution is changed by DMEM culture medium with 50 mu g/mL AC115619-22AA every day;

(3) the 400. Mu.g/mL AC115619-22AA treated group was added with 100. Mu.L of an aqueous solution of AC115619-22AA + 1900.0. Mu.L DMEM medium to make the AC115619-22AA content in the culture system 400. Mu.g/mL, placed at 37 ℃ and 5% CO ₂ Was cultured in an incubator of (1) for 1 week, and the medium was changed with DMEM medium containing 400. Mu.g/mL AC115619-22AA daily. The colony formation was observed using crystal violet staining.

The 0 μ g/mL AC115619-22AA treatment group finally forms 125.0 +/-12.2 effective clones, the 50 μ g/mL AC115619-22AA treatment group finally forms 75.6 +/-6.2 effective clones, and the 400 μ g/mL AC115619-22AA treatment group forms 0 effective clone. The AC115619-22AA is prompted to inhibit the cloning formation of the hepatoma cells.

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

The result is shown in figure 7, the AC115619-22AA polypeptide with the concentration of 400 mu g/mL effectively inhibits the clonogenic capacity of hepatoma carcinoma cells, which indicates that the AC115619-22AA polypeptide has anticancer activity.

4. Detection of induction effect of AC115619-22AA polypeptide on liver cancer cell Apoptosis by RealTime Glo Annexin V Apoptosis Assay real-time Apoptosis monitoring kit

The specific steps of detecting the induction effect of AC115619-22AA on the apoptosis of the liver cancer cells by using a real-time apoptosis monitoring kit (Promega corporation (Beijing) biotechnology, inc., product number JA 1000) are as follows:

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

2) HUH7 liver cancer cells are planted in a 96-well cell culture plate at a density of 5000 cells/well, the final liquid amount of each well is 200 mu L DMEM medium, after the cells are attached to the wall, 6 treatment groups are set in an experiment, and 5 wells are arranged in each treatment group:

(1) adding 200 μ L DMEM medium to the 0 μ g/mL AC115619-22AA treated group, standing at 37 deg.C, 5% ₂ Culturing for 72 hours in a constant-temperature incubator;

(2) 50 μ g/mL AC115619-22AA treatment group was added with 1.25 μ 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, the mixture is subjected to 37 ℃ and 5% CO ₂ Culturing for 72 hours in a constant-temperature incubator;

(3) the 100. Mu.g/mL AC115619-22AA treated group was added with 2.5. Mu.L of an aqueous solution of AC115619-22AA + 197.5. Mu.L DMEM medium to make the AC115619-22AA content in the culture system 100. Mu.g/mL, placed at 37 ℃ and 5% CO ₂ Culturing for 72 hours in a constant-temperature incubator;

(4) adding 5.0 μ L of aqueous solution of AC115619-22AA and 195.0 μ L of DMEM medium into the 200 μ g/mL AC115619-22AA treated group to make the content of AC115619-22AA in the culture system be 200 μ g/mL, placing in a container37℃、5％CO ₂ Culturing for 72 hours in a constant-temperature incubator;

(5) the 400. Mu.g/mL AC115619-22AA treated group was added with 10. Mu.L of an aqueous solution of AC115619-22AA plus 190.0. Mu.L of LDMEM medium to make the AC115619-22AA content in the culture system 400. Mu.g/mL, placed at 37 ℃ and 5% CO ₂ The culture was carried out in a constant temperature incubator for 72 hours.

The apoptotic signal was detected every 12h using a bioluminescent detector (Spark 10M, product number) from imperial ken trade limited.

The result is shown in figure 8, the liver cancer cell apoptosis can be caused at 12h by the treatment of AC115619-22AA with the concentration of 50 mug/mL, and the cell apoptosis is more obvious with the increase of the concentration of AC115619-22AA.

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

Claims (9)

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

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

3. A drug having an activity of inhibiting 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. The medicament of claim 3, wherein: the tumor is liver cancer.

5. The medicament of claim 3 or 4, wherein the medicament has at least one of the following properties:

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

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

6. Use of the polypeptide of claim 1 for the preparation of a product having tumor cell inhibitory or anti-tumor activity.

7. The use of a pharmaceutically acceptable salt according to claim 2 in the preparation of a product having tumor cell inhibitory or anti-tumor activity.

8. The use of claim 6 or 7, wherein the tumor cell is a hepatoma cell.

9. A biomaterial, characterized in that it is any one of the following:

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

c2 An expression cassette comprising the nucleic acid molecule according to C1);

c3 A recombinant vector containing the nucleic acid molecule according to C1) or a recombinant vector containing the expression cassette according to C2).

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