A polypeptide SCTR-peptide and its application

Abstract

The present invention discloses a polypeptide SCTR-peptide and its application, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1. The polypeptide of the present invention is derived from SCTR, can target β-arrestin 2, and specifically inhibit the interaction between SCTR and β-arrestin 2, thereby weakening the ability of gastric cancer cells to proliferate, migrate and invade, and achieving the purpose of inhibiting the occurrence and development of gastric cancer. The polypeptide SCTR-peptide of the present invention has application potential in the preparation of drugs for inhibiting the progression of gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a polypeptide SCTR-peptide and an application thereof. Background Art

[0002] SCTR (secretin receptor) is a G protein-coupled receptor (GPCR) containing 440 amino acids. It belongs to the glucagon-VIP-secretin receptor family. It is widely expressed in tissues such as the stomach, duodenum, pancreas, and small intestine. It can stimulate the pancreas and liver to secrete fluid and delay gastric emptying.

[0003] Secretin (also known as secretin) is a classic gut hormone composed of 27 amino acids, mainly found in the upper part of the small intestine. The binding of secretin to its receptor SCTR leads to the activation of the intracellular secondary messenger system, triggering cellular processes. Like all members of the GPCR B family, the secretin receptor activates AC through the Gs protein, thereby accumulating cAMP and activating PKA. In addition, the secretin receptor is also coupled to the Gq unit. When its ligand is activated, SCTR signals through Gs by increasing intracellular cAMP and stimulating calcium mobilization through Gq proteins, and the process of signal transduction is regulated by GRKs and β-arrestin. The receptor and ligand are endocytosed to allow receptor turnover or degradation. Regarding the role of SCTR in cancer, studies have shown that SCTR is overexpressed in various endocrine tumor cells and plays a role in the proliferation of pheochromocytoma and adrenocortical carcinoma cells. At present, there are few studies on the relationship between SCTR and gastric cancer.

[0004] Considering the key role of β-arrestin 2 in the occurrence and development of various cancers, and the fact that SCTR is highly expressed in the stomach and regulated by β-arrestin 2, we speculate that if we can selectively interfere with the interaction between β-arrestin 2 and SCTR, it may inhibit the progression of gastric cancer. Developing peptide drugs that inhibit the interaction between β-arrestin 2 and SCTR may have a good effect in inhibiting the progression of gastric cancer. However, there is still a lack of relevant research results in the existing technology. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a polypeptide SCTR-peptide and its application. The polypeptide SCTR-peptide can target β-arrestin 2 and specifically inhibit the interaction between SCTR and β-arrestin 2, thereby inhibiting the occurrence and development of gastric cancer.

[0006] The present invention is achieved through the following technical solutions:

[0007] A polypeptide SCTR-peptide, whose amino acid sequence is shown as SEQ ID NO.1.

[0008] The nucleic acid molecule encoding the polypeptide SCTR-peptide has a nucleotide sequence as shown in SEQ ID NO.2.

[0009] An expression cassette, recombinant vector or recombinant bacteria containing the above nucleic acid molecule.

[0010] Use of the above polypeptide SCTR-peptide, the above nucleic acid molecule or the above expression box, recombinant vector or recombinant bacteria in the preparation of drugs for treating diseases mediated by SCTR-peptide.

[0011] Preferably, the SCTR-peptide-mediated disease is gastric cancer.

[0012] Preferably, the polypeptide SCTR-peptide specifically inhibits the interaction between SCTR and β-arrestin 2 by targeting β-arrestin 2, thereby weakening the ability of gastric cancer cells to proliferate, migrate and invade, thereby inhibiting the occurrence and development of gastric cancer.

[0013] Preferably, the drug is in any pharmaceutically therapeutically acceptable dosage form.

[0014] A pharmaceutical composition for treating gastric cancer, comprising the above-mentioned polypeptide SCTR-peptide.

[0015] Preferably, a pharmaceutically acceptable drug delivery carrier is also included.

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention designs a polypeptide SCTR-peptide based on the carboxyl terminal region where SCTR may interact with β-arrestin 2. The polypeptide is derived from SCTR and can replace SCTR to interact with β-arrestin 2, reduce the recruitment of β-arrestin 2 to SCTR, and then reduce endocytosis and desensitization, weaken the ability of gastric cancer cells to proliferate, migrate and invade, and achieve the purpose of inhibiting the occurrence and development of gastric cancer. Therefore, the polypeptide SCTR-peptide of the present invention has application potential in the preparation of drugs for inhibiting the progression of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The EdU experiment in Example 2 was used to detect the proliferation ability of gastric cancer cells;

[0019] Figure 2 The scratch test in Example 2 was used to detect the migration ability of gastric cancer cells;

[0020] Figure 3 The transwell experiment in Example 2 was used to detect the migration and invasion ability of gastric cancer cells. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The given embodiments are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0022] The quantitative experiments in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0023] The experimental methods in the following examples, unless otherwise specified, are conventional molecular biology methods.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] Example 1 Acquisition of SCTR-peptide overexpression virus

[0026] This example describes the process of obtaining a lentivirus that overexpresses the SCTR-peptide active polypeptide. The specific steps are as follows:

[0027] The SCTR carboxyl-terminal polypeptide coding sequence was obtained by synthetic gene, cloned into the lentiviral vector pcAAV-CMV-EGFP-3xFLAG-WPRE, and packaged to obtain a lentivirus overexpressing SCTR-peptide, pcAAV-CMV-SCTR-EGFP-3xFLAG-WPRE. The lentivirus-expressed polypeptide had an EGFP tag and a FLAG tag at the C-terminus (synthesized by Heyuan Biotechnology (Shanghai) Co., Ltd.).

[0028] The amino acid sequence of the polypeptide SCTR-peptide is shown in SEQ ID NO.1, which consists of 49 amino acid residues and constitutes a region capable of interfering with the function of the protein β-arrestin 2. The nucleotide sequence of the gene encoding the polypeptide SCTR-peptide is shown in SEQ ID NO.2, which is 147 bp in length.

[0029] Example 2 Application research of polypeptide SCTR-peptide

[0030] 1. Cell culture

[0031] (1) Cell recovery

[0032] After taking out the cell cryopreservation tube, quickly place it in a 37℃ water bath to thaw, then transfer the cell suspension into a new EP tube in the clean bench, centrifuge at 4000rpm for 3min and discard the supernatant, add 1mL of culture medium to resuspend the cells and transfer them to the culture dish, then add an appropriate amount of culture medium to make up, shake well and move to the cell culture incubator.

[0033] (2) Cell passaging

[0034] When the cell density in the culture medium reaches 80% to 90%, the cells can be subcultured. Move the culture dish to the clean bench, discard the old culture medium, wash twice with PBS, add 1 mL of trypsin digestion solution for digestion, determine the digestion time according to the trypsin instructions and experience, discard the digestion solution after completion, add 2 mL of culture medium and mix by pipetting. Then distribute the cells to two new culture dishes, gently shake the culture dishes to evenly distribute the cells, and return them to the incubator to continue culturing.

[0035] (3) Cell cryopreservation

[0036] Digest and centrifuge the cells according to the steps, discard the supernatant, take 1 mL of freezing solution to resuspend the cells, then transfer to a freezing tube and store in a -80°C refrigerator.

[0037] 2. Lentivirus transfection and screening

[0038] Taking a 6-well plate as an example, the cells were counted and plated one day before transfection (2-3×10 5 cells / six-well plate) so that the cell density reaches 40% to 50% when infected with the virus the next day. In order to obtain satisfactory transfection efficiency, a preliminary experiment was first conducted to determine the appropriate Polybrene concentration and MOI value. Finally, the gastric cancer cell SGC-7901 was transfected with a Polybrene concentration of 6μg / mL and an MOI value of 10. Add the virus stock solution and Polybrene to the 6-well plate according to the above concentration and virus dosage. After 12 to 24 hours of viral infection, observe the cell state and replace fresh culture medium if necessary. It needs to be replaced every 48 hours thereafter. After 72 to 96 hours of viral infection, observe the efficiency of viral infection of the target cells under a fluorescence microscope. Select cells that have been successfully infected with the virus for the next step of screening, add an appropriate amount of puromycin and continue to culture for 3 to 5 days, and select cells with good growth status for subsequent functional tests and other related studies.

[0039] 3. Cell function test

[0040] (1) Edu experiment

[0041] After digestion, resuspending and counting, the cells were counted at 6×10 3 / well was inoculated into a 96-well plate and placed in an incubator for 24 hours. The Edu stock solution was diluted with complete culture medium to prepare a labeling solution with a concentration of 20μM. 200μL of labeling solution was added to each well and incubated in an incubator for 2 hours. All subsequent steps must be operated in the dark. The Edu labeling solution was discarded, 100μL of 4% paraformaldehyde was added, and it was discarded after fixation for 15 minutes. 100μL of washing solution (PBS+3% BSA) was added, and it was washed on a shaker for 5 minutes 3 times. Then 100μL of permeabilization solution (PBS+0.3% TritonX-100) was added, and it was permeabilized at room temperature for 15 minutes. 50μL of reaction working solution was added to each well. The working solution was prepared according to the instructions in the Biyuntian EdU-555 kit, incubated in the dark for 30 minutes, and washed with washing solution for 5 minutes 3 times. Add 100 μL of 10 μg / mL Hoechst 33342 staining solution, incubate for 10 min in the dark, wash 3 times for 5 min with washing solution, add anti-quenching reagent, store in a 4°C refrigerator in the dark, or immediately take pictures using a fluorescence microscope in a dark room.

[0042] (2) Scratch test

[0043] Draw a horizontal line on the back of the 6-well plate and digest, resuspend and count the cells. 5 / well inoculated into 6-well plates and cultured in an incubator. When the cell confluence in the 6-well plate reaches 80%, sterilize the ruler and place it on the 6-well plate. Use a 200μL pipette tip to draw two parallel horizontal lines on the bottom of the plate perpendicular to the horizontal lines on the back of the plate. Wash twice with PBS, place under a microscope and take pictures, replace the culture medium with serum-free culture medium, continue to culture for 24 hours and take pictures again. Note that the same position should be recorded in the pictures.

[0044] (3) Transwell experiment

[0045] Transwell experiments are divided into migration experiments and invasion experiments. For invasion experiments, the bottom of the chamber needs to be coated with matrix gel in advance. Specifically, take out the matrix gel and dilute it with serum-free medium: matrix gel = 9:1, add 100μL of diluted matrix gel to the bottom of the chamber, and put it in the incubator for 2-3 hours to wait for the matrix gel to solidify. The rest of the steps are the same as the migration experiment, as follows: After digestion, resuspending, and counting, the cells are counted at 1×10 4 10 cells / well were inoculated into the upper chamber of the chamber in a 24-well plate, with a total volume of 200 μL in each well of the upper chamber, and then 800 μL of complete culture medium containing 20% ​​FBS was added to the lower chamber of the chamber, which was placed in an incubator for continued culture.

[0046] After culturing for 24 h in the migration experiment or 48 h in the invasion experiment, the chamber was removed with tweezers and the liquid in the chamber was gently shaken off. The chamber was gently rinsed in PBS solution, and then 4% paraformaldehyde was added to the bottom of the chamber for fixation for 20 min. The chamber was rinsed twice with PBS, and then 1‰ crystal violet solution was added for staining for 15 min. Finally, the chamber was removed and rinsed repeatedly in ddH2O for several times. After drying, it was placed under an inverted microscope for photography.

[0047] 4. Experimental Grouping

[0048] NC group (control group): SGC-7901 cells not transfected with lentivirus.

[0049] SCTR-peptide group (experimental group): SGC-7901 cells transfected with the lentivirus described in Example 1.

[0050] 5. Experimental results

[0051] The results of the EdU experiment are as follows Figure 1 As shown, compared with the NC group, the percentage of Edu-positive cells in the gastric cancer cells transfected with SCTR-peptide was significantly reduced ( ** P<0.01)

[0052] The scratch test results are as follows Figure 2 As shown, compared with the NC group, the percentage of gastric cancer cell migration area in the SCTR-peptide transfected group was significantly reduced at 24 h ( *** P<0.001)

[0053] Transwell experiment results Figure 3 As shown, compared with the NC group, the average number of gastric cancer cells per field of view in the SCTR-peptide transfected group was significantly reduced ( ** P<0.01).

[0054] From the above experimental results, it can be seen that the polypeptide SCTR-peptide of the present invention can significantly inhibit the proliferation, migration and invasion of gastric cancer cells.

[0055] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A polypeptide SCTR-peptide, characterized in that: The amino acid sequence of the polypeptide SCTR-peptide is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the polypeptide SCTR-peptide according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

3. An expression cassette, recombinant vector or recombinant bacterium containing the nucleic acid molecule according to claim 2.

4. Use of the polypeptide SCTR-peptide according to claim 1, the nucleic acid molecule according to claim 2 or the expression cassette, recombinant vector or recombinant bacteria according to claim 3 in the preparation of a drug for treating gastric cancer.

5. The use according to claim 4, characterized in that: The drug is in any therapeutically acceptable dosage form.

6. A pharmaceutical composition for treating gastric cancer, characterized in that: The invention comprises the polypeptide SCTR-peptide according to claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that Also included are pharmaceutically acceptable delivery vehicles.

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