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

An anti-cancer peptide AC115619-22AA encoded by lncRNA and its application

Technical field

The invention belongs to the technical field of polypeptide drugs in biochemistry, and specifically relates to an anti-cancer peptide AC115619-22AA encoded by lncRNA and its application.

Background technique

The overall effect of patients with intermediate and advanced liver cancer is poor, with a mortality rate as high as 80%, a median survival period of less than 1 year, and a 5-year survival rate of less than 20%. Conventional liver cancer treatment drugs are mostly chemotherapy drugs and small molecule targeted drugs. These drugs have serious side effects and are prone to drug resistance. Although drugs such as immune checkpoint inhibitors have clear effects, they are limited by the patient's immune microenvironment and the response rate does not exceed 30%. Therefore, it is necessary to develop new drugs to treat liver cancer.

Long non-coding RNA (lncRNA) is a non-protein-coding RNA consisting of more than 200 bases. The traditional theory is that lncRNA does not have the ability to encode amino acids, but in recent years, more and more evidence has confirmed that lncRNA is not completely incapable of encoding amino acids. Although it cannot encode large-molecule proteins, many lncRNAs can pass through small open reading frames (small open reading frames). frame, sORF) encodes tiny polypeptide segments of less than 100 amino acids. The polypeptides encoded by these lncRNAs also play an important role in the biological processes of tumors. For example, some lncRNA-encoded polypeptides may inhibit the occurrence and development of tumors. This type of peptides can be used as anti-cancer peptide drugs. By supplementing these peptides, they can inhibit tumors. Therefore, identifying and utilizing the anti-cancer peptides encoded by these lncRNAs is of great clinical significance for the prevention and treatment of tumors.

Contents of the invention

The technical problem to be solved by the present invention is how to obtain these lncRNAs that can encode polypeptides that inhibit tumor growth.

In order to solve the above problems, the present invention provides a polypeptide AC115619-22AA encoded by lncRNA-AC115619, and provides its application in preparing drugs for treating liver cancer.

The present invention first provides a polypeptide.

The polypeptide provided by the present invention is a polypeptide whose amino acid sequence is SEQ ID No. 2 in the sequence listing.

The present invention also provides pharmaceutically acceptable salts of the above polypeptides.

Pharmaceutically acceptable salts of polypeptides of the present invention include acetate, lactobionate, benzenesulfonate, laurate, benzoate, and malate. (malate), bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, boron Borate, methylbromide, bromide, methylnitrate, calciumedetate, methylsulfate, camsylate, viscosity Mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine ), citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, ethylenediamine Edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate (pantothenate), fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate , salicylate, glutamate, stearate, glycollylarsanilate, sulfate, hexylresorcinate , subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate ), hydroxynaphthoate, teoclate, iodide, tosylate, triethiodide, lactate, valerate (valerate) etc. Depending on the use, pharmaceutical salts may be composed of cations such as sodium, potassium, aluminum, calcium, lithium, magnesium and zinc, bismuth It can also be formed from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine (ornithine), choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, Prunus It is formed from procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane and tetramethylammonium hydroxide. 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, acidic salts such as hydrochloride, hydrobromide, acetate, pamoate, etc. can be used as dosage forms; In the presence of an acidic group (such as -COOH) or alcohol group, pharmaceutically acceptable esters such as acetate, maleate, pivaloyloxymethyl, etc., As well as esters known in the literature to improve solubility and hydrolyzability can be used as sustained release and prodrug formulations.

The present invention also provides drugs that have tumor cell inhibition activity or anti-tumor activity, and the drugs contain the polypeptides or pharmaceutically acceptable salts described above.

In practical applications, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be directly administered to a patient as a drug, or mixed with a suitable carrier or excipient and then administered to the patient. The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Preferred among these are water-soluble carrier materials. These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Oral tablets, suppositories, freeze-dried powder injections, etc. Among them, the suppository can be a vaginal suppository, a vaginal ring, or an ointment, cream or gel suitable for vaginal application. It can be ordinary preparations, sustained-release preparations, controlled-release preparations and various particulate drug delivery systems. To formulate unit dosage forms into tablets, a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbing agents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and adhesives, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia glue, gelatin slurry, sodium carboxymethyl cellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch, alginate, agar powder, fucoid starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid esters, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promotion Agents, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bi-layer and multi-layer tablets. To formulate unit dosage forms into pills, a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbers such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, and gelatin. , ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc. To formulate a unit dosage form as a suppository, a wide variety of carriers known in the art may be used. Examples of the carrier include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like. In order to formulate unit dosage forms into injection preparations, such as solutions, emulsions, lyophilized powder injections and suspensions, all diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation. In addition, conventional co-solvents, buffers, pH adjusters, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparations.

The above dosage forms can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; oral administration, such as rectum, vagina and sublingual; respiratory tract administration, such as nasal cavity ;Mucosal administration. The above route of administration is preferably injection.

Among the above-mentioned drugs, the tumor is liver cancer.

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

A1. Application in preparing products that inhibit tumor cell activity;

A2. Application in preparing products with anti-tumor activity.

The present invention also provides the use of the above-mentioned polypeptide in preparing products with tumor cell inhibition activity or anti-tumor activity.

The use of the above pharmaceutical salts in the preparation of products with tumor cell inhibition activity or anti-tumor activity also falls within the scope of protection claimed by the present invention.

In the above application, the tumor cells are liver cancer cells.

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

C1) A nucleic acid molecule encoding the polypeptide of claim 1;

C2) an expression cassette containing the nucleic acid molecule described in C1);

C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2).

The present invention provides a polypeptide AC115619-22AA encoded by lncRNA-AC115619, and studies its application in preparing drugs for treating liver cancer. After being dissolved in ultrapure water, the polypeptide can effectively inhibit the characteristics of cancer stem cells, inhibit the proliferation and migration of liver cancer cells, and induce apoptosis of liver cancer cells. Therefore, the polypeptide AC115619-22AA is an anti-cancer peptide with liver cancer inhibitory effect and can be a candidate component or material basis for drugs to treat liver cancer.

Description of the drawings

Figure 1 shows the identification of the synthetic AC115619-22AA anti-cancer peptide by high-performance liquid chromatography.

Figure 2 shows the identification of the synthetic AC115619-22AA anti-cancer peptide by liquid chromatography-mass spectrometry.

Figure 3 shows the uptake of AC115619-22AA polypeptide by liver cancer cells detected by immunofluorescence.

Figure 4 shows that the AC115619-22AA anti-cancer peptide effectively inhibits the spheroidization ability of liver cancer cells.

Figure 5 shows the inhibitory effect of AC115619-22AA anti-cancer peptide at different concentrations on liver cancer cells.

Figure 6 shows the inhibitory effect of 400 μg/mL AC115619-22AA anti-cancer peptide on liver cancer cells under different treatment time conditions.

Figure 7 shows the AC115619-22AA anti-cancer peptide effectively inhibits the clonogenic ability of liver cancer cells.

Figure 8 shows the induction of apoptosis of liver cancer cells by different concentrations of AC115619-22AA anti-cancer peptide at 12 hours.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Quantitative experiments in the following examples were repeated three times unless otherwise specified.

Example 1. Artificial synthesis of AC115619-22AA anti-cancer peptide

lncRNA-AC115619 (ENST00000567376.2) is a lncRNA located on human chromosome 2. The specific chromosome coordinates are Chromosome 2:20,999,313-21,000,917, and the length is 1605 bases. This lncRNA is a liver-specific lncRNA that is only expressed in the liver, and the lncRNA shows low expression in liver cancer cells.

Through ribosome sequencing and analysis using the ORFfinder database developed by the National Center for Biotechnology Information, a 66-base sORF region at bases 471 to 536 of this lncRNA was identified. This sORF region was first confirmed to encode a polypeptide. . The nucleotide sequence of this sORF is ATGGCAATACGGACCCCAGATGTCTCCCTGGAGCTCTGCCAGGATCCATTCAAGGCCAAGAAAGTA (sequence 1 in the sequence listing). The translated polypeptide contains 22 amino acids, and its amino acid sequence is: Met-Ala-Ile-Arg-Thr-Pro-Asp-Val-Ser-Leu -Glu-Leu-Cys-Gln-Asp-Pro-Phe-Lys-Al a-Lys-Lys-Val(Methionine-Alanine-Isoleucine-Arginine-Threonine-Proline Acid-aspartic acid-valine-threonine-leucine-glutamic acid-leucine-cysteine-glutamine-aspartic acid-proline-phenylalanine- Lysine-alanine-lysine-lysine-valine), the amino acid sequence is sequence 2 in the sequence listing, and the molecular weight is: 2488.7KDa. This peptide is called AC115619-22AA polypeptide or AC115619-22AA.

Based on the known amino acid sequence of AC115619-22AA, the peptide was artificially synthesized using solid-phase chemical synthesis. High-performance liquid chromatography (results shown in Figure 1) and liquid chromatography-mass spectrometry (results shown in Figure 2) were subsequently used to identify the synthesized AC115619-22AA.

The results show that the product purity of AC115619-22AA is >95% and its water solubility is good. It indicates that the AC115619-22AA polypeptide is synthesized correctly.

Example 2. Uptake of fluorescein isothiocyanate FITC combined with AC115619-22AA polypeptide by cells

When performing peptide synthesis, fluorescein isothiocyanate FITC was labeled to AC115619-22AA. HUH7 liver cancer cells (purchased from Wuhan Pronosai Life Technology Co., Ltd., product number: CL-0120) were seeded in a 6-well plate at a density of 20,000 cells/well and continued to be cultured routinely for 24 hours. Then, FITC-labeled AC115619-22AA polypeptide was added to DMEM culture medium (Wuhan Pronosai Life Technology Co., Ltd., Cat. No.: PM150210B) at a concentration of 400 μg/mL, and culture was continued for 4 hours. The results are shown in Figure 3. Immunofluorescence detection found that AC115619-22AA can be absorbed into the cytoplasm of liver cancer cells, indicating that the peptide is well absorbed by cells, can penetrate cell membranes, and has medicinal value.

Example 3. Study on the inhibitory effect of AC115619-22AA polypeptide on the tumor stem cell properties of liver cancer cells

The effect of AC115619-22AA on the tumor stem cell properties of liver cancer cells was observed using cell spheroidization assay. The experiment was repeated three times, and the specific steps for each repetition were as follows:

HUH7 liver cancer cells (purchased from Wuhan Punosai Life Technology Co., Ltd., product number: CL-0120) were planted in an ultra-low adsorption 6-well cell culture plate at a density of 10,000 cells/well, and 3 mL of DMEM medium was added for the experiment. There are two treatment groups: a polypeptide treatment group and a control group. AC115619-22AA aqueous solution was added to the peptide treatment group to make the final concentration 400 μg/mL, and the same amount of aqueous solution (0 μg/mL AC115619-22AA) was added to the control group. Five wells in each treatment group were cultured in a constant temperature incubator at 37°C and 5% _CO2 . After continuous culture for 1 week, the ball formation was observed under a microscope.

Experimental results show that treatment of cells with 400 μg/mL AC115619-22AA can effectively inhibit the sphere-forming ability of HUH7 cells. The cell sphere-forming ability is weakened and the sphere size is reduced (Figure 4), suggesting that AC115619-22AA inhibits the stem cell properties of liver cancer cells. .

Example 4. AC115619-22AA polypeptide inhibits the proliferation of liver cancer cells and induces apoptosis

1. Study on the concentration of AC115619-22AA polypeptide in inhibiting liver cancer cells

The CCK-8 kit (purchased from the company Shanghai Yisheng Biotechnology Co., Ltd., product number: 40203ES60) was used to analyze the inhibitory effect of AC115619-22AA polypeptide on the proliferation of liver cancer cells. The experiment was repeated three times, and the specific steps for each repetition were as follows:

1) Prepare AC115619-22AA aqueous solution: Use sterile enzyme-free water as the solvent and AC115619-22AA as the solute to prepare an AC115619-22AA aqueous solution (concentration: 8.0 mg/mL).

2) Plant HUH7 liver cancer cells into a 96-well cell culture plate at a density of 5,000 cells/well. The final liquid volume in each well is 200 μL DMEM culture medium. After the cells adhere, the experiment is divided into 6 treatment groups, with 5 cells in each treatment group. hole:

① Add 200 μL DMEM medium to the 0 μg/mL AC115619-22AA treatment group and place it in a constant temperature incubator at 37°C and 5% _CO2 for 48 hours;

② In the 50 μg/mL AC115619-22AA treatment group, add 1.25 μL AC115619-22AA aqueous solution + 198.75 μL DMEM culture medium to make the content of AC115619-22AA in the culture system 50 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 48 hours;

③Add 2.5 μL AC115619-22AA aqueous solution + 197.5 μL DMEM medium to the 100 μg/mL AC115619-22AA treatment group to make the AC115619-22AA content in the culture system 100 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 48 hours;

④Add 5.0 μL AC115619-22AA aqueous solution + 195.0 μL DMEM culture medium to the 200 μg/mL AC115619-22AA treatment group to make the AC115619-22AA content in the culture system 200 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 48 hours;

⑤Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM culture medium to the 400 μg/mL AC115619-22AA treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 48 hours.

3) Use 1:100 CCK-8 reagent to detect changes in cell activity in each treatment group, and use a microplate reader to detect the absorbance at a wavelength of 450 nm to reflect cell activity. Cell inhibition rate = 1-(absorbance at detection time point/absorbance at 0 h time point)*100%.

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

The CCK-8 kit (purchased from the company Shanghai Yisheng Biotechnology Co., Ltd., product number: 40203ES60) was used to analyze the inhibitory effect of AC115619-22AA polypeptide on the proliferation of liver cancer cells. The experiment was repeated three times, and the specific steps for each repetition were as follows:

1) Prepare AC115619-22AA aqueous solution: Use sterile enzyme-free water as the solvent and AC115619-22AA as the solute to prepare an AC115619-22AA aqueous solution (concentration: 8.0 mg/mL).

2) Plant HUH7 liver cancer cells in a 96-well cell culture plate at a density of 5,000 cells/well. The final liquid content in each well is 200 μL DMEM culture medium. After the cells adhere, the experiment is divided into 4 treatment groups:

①Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM culture medium to the 0-hour treatment group, place it in a constant-temperature incubator at 37°C and 5% _CO2 , and incubate for 0 hours before testing;

②Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM medium to the 12-hour treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL. Place it in a constant-temperature incubator at 37°C and 5% _CO2 and then detect it after 12 hours of cultivation. ;

③Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM culture medium to the 24-hour treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL. Place it in a constant temperature incubator at 37°C and 5% _CO2 and then detect it after 24 hours of cultivation. ;

④Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM culture medium to the 48-hour treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL. Place it in a constant-temperature incubator at 37°C and 5% _CO2 and then detect it after 48 hours. ;

⑤Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM culture medium to the 72-hour treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL. Place it in a constant-temperature incubator at 37°C and 5% _CO2 and then detect it after 72 hours of cultivation. ;

5 wells per treatment group.

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

The results are shown in Figure 5 and Figure 6. AC115619-22AA at a concentration of 100 μg/mL can show a relatively obvious inhibitory effect on HUH7 liver cancer cells. As the concentration of AC115619-22AA polypeptide increases, the survival rate of HUH7 liver cancer cells gradually decreases. ; Using 400 μg/mL AC115619-22AA peptide to treat HUH7 liver cancer cells showed an inhibitory effect on HUH7 liver cancer cells at 24 hours. Therefore, the AC115619-22AA polypeptide has anticancer activity.

3. Clone formation method to analyze the inhibitory effect of AC115619-22AA polypeptide on the proliferation of liver cancer cells

The experiment was repeated three times, and the specific steps for each repetition were as follows:

1) Prepare AC115619-22AA aqueous solution: Use sterile enzyme-free water as the solvent and AC115619-22AA as the solute to prepare an AC115619-22AA aqueous solution (concentration: 8.0 mg/mL).

2) Plant HUH7 liver cancer cells (purchased from Wuhan Punosai Life Technology Co., Ltd., product number: CL-0120) at a density of 1000 cells/well in a 6-well cell culture plate, add 3 mL of DMEM culture medium, and allow the cells to adhere. Finally, the experiment was set up with 3 treatment groups:

①Add 2000μg DMEM culture medium to the 0μg/mL AC115619-22AA treatment group and place it in a constant temperature incubator at 37°C and 5% _CO2 for 1 week. Change the medium with DMEM medium every day;

② Add 12.5 μL AC115619-22AA aqueous solution + 1975.5 μL DMEM culture medium to the 50 μg/mL AC115619-22AA treatment group to make the AC115619-22AA content in the culture system 50 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . For 1 week, replace the medium with DMEM medium with 50 μg/mL AC115619-22AA every day;

③Add 100 μL AC115619-22AA aqueous solution + 1900.0 μL DMEM culture medium to the 400 μg/mL AC115619-22AA treatment group to make the AC115619-22AA content in the culture system 400 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 for culture 1 For weeks, the medium was replaced with DMEM medium containing 400 μg/mL AC115619-22AA every day. Use crystal violet staining to observe colony formation.

The 0 μg/mL AC115619-22AA treatment group finally formed 125.0±12.2 effective clones, the 50 μg/mL AC115619-22AA treatment group finally formed 75.6±6.2 effective clones, and the 400 μg/mL AC115619-22AA treatment group formed 0 effective clones. It is suggested that AC115619-22AA inhibits the clonogenic formation of liver cancer cells.

Among them, the DMEM+50 μg/mL medium is a liquid obtained by adding the above-mentioned AC115619-22AA aqueous solution to the DMEM medium. The content of AC115619-22AA in the DMEM+50 μg/mL medium is 50 μg/mL. DMEM+400μg/mL medium is a liquid obtained by adding the above-mentioned AC115619-22AA aqueous solution to DMEM medium. The content of AC115619-22AA in DMEM+400μg/mL medium is 400μg/mL.

The results are shown in Figure 7. AC115619-22AA polypeptide at a concentration of 400 μg/mL effectively inhibited the clonogenic ability of liver cancer cells, indicating that AC115619-22AA polypeptide has anti-cancer activity.

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

The specific steps for detecting the apoptosis-inducing effect of AC115619-22AA on liver cancer cells using a real-time apoptosis monitoring kit (Promega (Beijing) Biotechnology Co., Ltd., Cat. No. JA1000) are as follows:

1) Preparation of AC115619-22AA aqueous solution: Use water as the solvent and AC115619-22AA as the solute to prepare an AC115619-22AA aqueous solution (concentration: 8.0 mg/mL).

2) Plant HUH7 liver cancer cells into a 96-well cell culture plate at a density of 5,000 cells/well. The final liquid volume in each well is 200 μL DMEM culture medium. After the cells adhere, the experiment is divided into 6 treatment groups, with 5 cells in each treatment group. hole:

① Add 200 μL DMEM culture medium to the 0 μg/mL AC115619-22AA treatment group and place it in a constant temperature incubator at 37°C and 5% _CO2 for 72 hours;

② In the 50 μg/mL AC115619-22AA treatment group, add 1.25 μL AC115619-22AA aqueous solution + 198.75 μL DMEM medium to make the AC115619-22AA content in the culture system 50 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 for 72 days. Hour;

③Add 2.5 μL AC115619-22AA aqueous solution + 197.5 μL DMEM medium to the 100 μg/mL AC115619-22AA treatment group to make the AC115619-22AA content in the culture system 100 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 72 hours;

④Add 5.0 μL AC115619-22AA aqueous solution + 195.0 μL DMEM culture medium to the 200 μg/mL AC115619-22AA treatment group to make the AC115619-22AA content in the culture system 200 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 72 hours;

⑤Add 10 μL AC115619-22AA aqueous solution + 190.0 μL DMEM culture medium to the 400 μg/mL AC115619-22AA treatment group to make the content of AC115619-22AA in the culture system 400 μg/mL, and place it in a constant temperature incubator at 37°C and 5% _CO2 . 72 hours.

A bioluminescence detector (Tecan (Shanghai) Trading Co., Ltd., Cat. No. Spark10M) was used to detect apoptosis signals every 12 hours.

The results are shown in Figure 8. Treatment with AC115619-22AA at a concentration of 50 μg/mL can cause apoptosis of liver cancer cells at 12 hours. As the concentration of AC115619-22AA increases, cell apoptosis becomes more obvious.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Claims (8)

1. A polypeptide, characterized in that: the polypeptide is a polypeptide whose amino acid sequence is SEQ ID No. 2 in the sequence listing. 2. A pharmaceutically acceptable salt of the polypeptide of claim 1. 3. A drug with tumor cell inhibition activity or anti-tumor activity, characterized in that the drug contains the polypeptide of claim 1 or the pharmaceutical salt of claim 2. 4. The medicine according to claim 3, characterized in that: the tumor is liver cancer. 5. The medicine according to claim 3 or 4, characterized in that the medicine has at least one of the following properties: A1. Application in preparing products that inhibit tumor cell activity; A2. Application in preparing products with anti-tumor activity. 6. Application of the polypeptide of claim 1 in the preparation of products with inhibitory activity or anti-tumor activity on tumor cells, where the tumor cells are liver cancer cells. 7. The medicinal salt according to claim 2 is used in the preparation of products with inhibitory activity or anti-tumor activity on tumor cells, where the tumor cells are liver cancer cells. 8. Biological material, characterized in that the biological material is any of the following: C1) A nucleic acid molecule encoding the polypeptide of claim 1; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2).