Application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer

Abstract

This invention discloses the application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer, belonging to the field of biomedical technology. This invention discovers that the small interfering RNA of SLC10A3 exerts an anti-tumor effect by inhibiting the proliferation, invasion, and migration of gastric cancer cells and promoting ferroptosis. Inhibiting endogenous SLC10A3 in the SGC-7901 gastric cancer cell line can inhibit the proliferation, invasion, and migration of gastric cancer cells and promote ferroptosis. Combining the small interfering RNA of SLC10A3 with a suitable carrier to form a drug, and introducing it into gastric tissue or the body, will have preventive and therapeutic effects on gastric cancer. It is hoped that the small interfering RNA of SLC10A3 can be combined with chitosan, cholesterol, liposomes, nanoparticles, etc., to form drug molecules for the prevention and treatment of gastric cancer via oral, intravenous, intramuscular, or direct intragastric injection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors in the world that seriously affect human health. Gastric cancer is characterized by high incidence, low early diagnosis rate, high recurrence rate, and high mortality rate. Therefore, finding new diagnostic biomarkers and therapeutic targets, and further exploring the mechanisms of its occurrence and development, are crucial for the diagnosis and treatment of gastric cancer.

[0003] Solute carriers (SLCs) are the second largest family of membrane proteins in the human body after G protein-coupled receptors. They can transport various substances such as carbohydrates, amino acids, vitamins, ions, nucleic acids, and drugs, and have been shown to be associated with various cancers, including liver cancer, pancreatic cancer, and breast cancer. However, there is still much room for research in gastric cancer. Members of the SLC superfamily can participate in various biological behaviors of tumors, such as invasion, proliferation, metastasis, apoptosis, and chemotherapy resistance.

[0004] The SLC10 family is an important member of the SLC transporter family, typically encoding proteins responsible for transporting various bile acids and steroid hormones. Previous studies have found that solute carrier family member 10, member 3 (SLC10A3), is an atypical member of the solute carrier family, and its specific biological function remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide the application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer, thereby addressing the problems existing in the prior art. This invention aims to identify or discover SLC family members highly expressed in gastric tissue that regulate the occurrence and development of gastric cancer, determine their key roles in the proliferation, invasion, and migration of gastric cancer cells, and apply their detection to the diagnosis and prevention of gastric cancer.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is the application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The second technical solution of the present invention is the application of SLC10A3 as a target in screening candidate drugs for the prevention and / or treatment of gastric cancer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The third technical solution of this invention is the application of SLC10A3 expression inhibitors, knockout reagents, or silencing reagents in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0009] The fourth technical solution of the present invention is a small interfering RNA of SLC10A3, the nucleotide sequence of which is shown in SEQ ID NO.6 or SEQ ID NO.7.

[0010] The fifth technical solution of the present invention is a drug for preventing and / or treating gastric cancer, comprising the small interfering RNA and a pharmaceutically acceptable carrier.

[0011] The sixth technical solution of this invention is the application of a reagent for detecting SLC10A3 expression levels in the preparation of products for gastric cancer diagnosis or prognostic assessment.

[0012] Based on the above technical solution, the present invention has the following technical effects: (1) The SLC10A3 provided by the present invention is significantly upregulated in human gastric cancer tissues and gastric cancer cell lines. Specific detection of this SLC10A3 can be used to diagnose and / or assess the prognosis of gastric cancer.

[0013] (2) The present invention also provides two small interfering RNAs of SLC10A3, which can inhibit the expression of SLC10A3 and play a role in inhibiting the proliferation, invasion and migration of gastric cancer cells, promoting ferroptosis and thus anti-tumor effects; the small interfering RNA of SLC10A3 is used as a drug and introduced into gastric tissue or the body, which will have preventive and therapeutic effects on gastric cancer. Attached Figure Description

[0014] Figure 1 To detect the expression level of SLC10A3 in gastric cancer cell lines after small interfering RNA transfection.

[0015] Figure 2 To investigate the effect of endogenous SLC10A3 on the proliferation of gastric cancer cells SGC-7901.

[0016] Figure 3 This image shows the effect of SLC10A3 small interfering RNA on the invasion and migration abilities of gastric cancer cells SGC-7901. In the image, A represents a representative image showing the reduced invasion and migration abilities of SGC-7901 gastric cancer cells after using SLC10A3 small interfering RNA; B shows the quantitative results of migration ability (a bar chart comparing the average cell count from 5 high-power fields with the control group); and C shows the quantitative results of invasion ability (a bar chart comparing the average cell count from 5 high-power fields with the control group).

[0017] Figure 4 This illustrates the effect of SLC10A3 small interfering RNA on ferroptosis in gastric cancer cells SGC-7901, as shown in Example 1. Here, A represents the iron ion level, and B represents the reactive oxygen species (ROS) level. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides the application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0020] In some specific implementations, the gastric cancer includes one or more of the following: self-elevating gastric cancer, localized ulcerative gastric cancer, invasive ulcerative gastric cancer, and diffuse invasive gastric cancer.

[0021] This invention also provides the application of SLC10A3 as a target in screening candidate drugs for the prevention and / or treatment of gastric cancer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0022] This invention also provides the use of SLC10A3 expression inhibitors, knockout agents, or silencing agents in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0023] This invention also provides a small interfering RNA of SLC10A3, the nucleotide sequence of which is shown in SEQ ID NO.6 or SEQ ID NO.7.

[0024] This invention also provides a medicament for the prevention and / or treatment of gastric cancer, comprising the small interfering RNA and a pharmaceutically acceptable vector.

[0025] In some specific implementations, the carrier includes chitosan, cholesterol, liposomes, or nanoparticles.

[0026] In some specific implementations, the drug exerts its therapeutic or preventive effect on gastric cancer by inhibiting the proliferation of gastric cancer cells, inhibiting the invasion and migration of gastric cancer cells, and promoting ferroptosis of gastric cancer cells.

[0027] This invention also provides the use of reagents for detecting SLC10A3 expression levels in the preparation of products for gastric cancer diagnosis or prognostic assessment. The SLC10A3 is upregulated in gastric cancer patients.

[0028] In some specific implementations, the product includes reagents, reagent kits, or chips.

[0029] In some specific embodiments, the product is a kit comprising primers for specifically amplifying SLC10A3 and primers for specifically detecting SLC10A3. The primers for specifically amplifying SLC10A3 include an upstream primer with the sequence shown in SEQ ID NO.2 and a downstream primer with the sequence shown in SEQ ID NO.3. The primers for specifically detecting SLC10A3 include an upstream primer with the sequence shown in SEQ ID NO.4 and a downstream primer with the sequence shown in SEQ ID NO.5.

[0030] The experiments of this invention show that knocking down SLC10A3 can inhibit the proliferation, migration, and invasion of gastric cancer cells in vitro, and increase the levels of total iron ions and reactive oxygen species (ROS), markers of ferroptosis in gastric cancer cells. Elucidating its mechanism of action is of great significance and has promising application prospects for developing diagnostic and therapeutic methods for gastric cancer related to members of the SLC10 family.

[0031] Example 1 On the UALCAN database website (http: / / ualcan.path.uab.edu), click the TCGA section on the main interface, enter the target gene name SLC10A3 in the core search area, and select the Stomach Adenocarcinoma (STAD) dataset from the "TCGA Dataset" drop-down menu. Click "Explore" to jump to the expression analysis interface.

[0032] The results showed that the transcript expression level of SLC10A3 was significantly increased in primary gastric cancer tissues compared with adjacent normal tissues (P<0.05), indicating that SLC10A3 is highly expressed in gastric cancer. Using the "Tumor grade" grouping method, the results showed that the expression level of SLC10A3 increased sequentially in Grade 1, Grade 2, and Grade 3 gastric cancer tissues, and was significantly higher than that in adjacent normal tissues. The difference between Grade 2 and Grade 3 was statistically significant (P<0.05), indicating that the upregulation of SLC10A3 expression is closely related to the malignancy grade of gastric cancer.

[0033] Detection of SLC10A3 knockdown effect in gastric cancer cell line SGC-7901 In this embodiment, the gastric cancer cell line SGC-7901 was cultured and cryopreserved under normal culture conditions. After cell counting, SGC-7901 cells were seeded into 6-well plates and transfected using Gencefe Super Trans liposomes. A control group and a small interfering RNA (SRNA) experimental group were established. The SRNA concentration was 10 μM, and the volume was 7.5 μL / well. Transfection was performed according to the kit instructions. Gencefe Super Trans liposomes were purchased from Jiangsu Gencefe Co., Ltd. SI-SLC10A3-2 targets the sequence starting at nucleotide 623 of the SLC10A3 sequence (SEQ ID NO. 6), and SI-SLC10A3-3 targets the sequence starting at nucleotide 212 (SEQ ID NO. 7). Six hours after transfection, the medium was replaced with normal medium, and the cells were cultured for another day. The medium was then aspirated, and Trizol was added to harvest the cells.

[0034] The sense strand sequence in the small interfering RNA that inhibits SLC10A3 synthesis is as follows: 5'-GGTGCAAAGTGGAACTCGATT-3' (SEQ ID NO. 6); 5'-GCTCTGTGATGGAGTTTGATT-3' (SEQ ID NO. 7).

[0035] Total RNA was extracted from gastric cancer cells using the Trizol reagent method, and the expression level of SLC10A3 was detected using real-time quantitative PCR. SLC10A3 was amplified by PCR, and the full-length nucleotide sequence was 1434 bp, as shown in SEQ ID NO. 1.

[0036]

[0037] The PCR reaction system for amplifying the full-length gene was 50 μL: 10.0 μL of 5×PS Buffer, 4.0 μL of dNTP Mix (2.5 mMeach), 1.0 μL of upstream amplification primer (10 µM), 1.0 μL of downstream amplification primer (10 µM), 1.0 μL of template (10 ng / µL), 0.5 μL of PrimeSTAR HS DNA polymerase, and double-distilled water to bring the volume to 50 μL. The PCR conditions were as follows: 95℃, 5 min, one cycle; 95℃, 30 sec; 56℃, 30 sec; 72℃, 1.5 min, for a total of 30 cycles; and a final extension at 72℃ for 7 min.

[0038] The PCR primers are designed as follows: Upstream primer: 5'-TGCTGGATATCTGCAGAATTCATGGTGTTAATGC-3′ (SEQ ID NO.2); Downstream primer: 5'-CTTGGTACCGAGCTCGGATCCTCAGGGAAC-3' (SEQ ID NO.3).

[0039] The expression level of SLC10A3 was detected by real-time quantitative PCR. The PCR reaction system was 20 μL: SYBRGreen Real-time PCR Master Mix 10.0 μL; Primer F 1.0 μL; Primer R 1.0 μL; cDNA 2.0 μL; triple-distilled water to make up to 20.0 μL.

[0040] qPCR conditions: 95℃, 30 sec, one cycle; 95℃, 5 sec; 60℃, 30 sec; 72℃, 1 min, for a total of 39 cycles; final extension at 72℃ for 10 min.

[0041] The PCR primers are designed as follows: The upstream primer for specific detection of the SLC10A3 fragment is 5′-GGGACCCTGCTGTTCATTG-3′ (SEQ ID NO.4); the downstream primer for specific detection of the SLC10A3 fragment is 5′-CACCAGTACGATGGGTAGCC-3′ (SEQ ID NO.5).

[0042] In this embodiment, the expression level of SLC10A3 in gastric cancer cell lines was detected using real-time quantitative PCR. The results are as follows: Figure 1 As shown, Figure 1The vertical axis represents the significant decrease in SLC10A3 expression level in the gastric cancer cell line SGC-7901 after the use of small interfering RNA, with the internal reference gene GAPDH (NM_014364) as the benchmark.

[0043] Example 2 Experiments on the inhibition of gastric cancer cell proliferation by small interfering RNA of SLC10A3 The gastric cancer cell line SGC-7901, treated with small interfering RNA from SLC10A3 in Example 1, was used as a cell model and seeded into 96-well plates (approximately 6 × 10⁶ cells). 3 After the cells adhered to the culture vessel, they were cultured for 1-5 days. After incubation in an incubator for different times, 100 μL of culture medium was discarded, and 10 μL of MTT (final concentration 500 μg / mL) was added. After culturing for another 4 hours, 150 μL of DMSO was added to dissolve the formazan particles, and the OD 490nm light absorbance was detected using an ELISA reader.

[0044] Experimental results are as follows Figure 2 As shown, the results indicate that inhibiting endogenous SLC10A3 can significantly suppress the proliferation of gastric cancer cells SGC-7901.

[0045] Example 3 SLC10A3's small interfering RNA inhibits the invasion and migration of gastric cancer cells. The gastric cancer cell line SGC-7901, treated with small interfering RNA from SLC10A3 in Example 1, was used as a cell model. The invasive ability of the cells was detected using the Transwell assay, the specific procedures of which are as follows: The upper surface of the bottom membrane of the Transwell chamber was coated with 50 mg / L Matrigel at a 1:10 dilution and incubated at 37°C. After treatment with SLC10A3 small interfering RNA, the cells were digested 12 hours later, washed once with PBS and once with serum-free medium, resuspended in serum-free medium, counted, and the concentration adjusted to 3 × 10⁻⁶ cells / mL. 5 / mL; Add 600-800μL of culture medium containing 20% ​​serum to the lower chamber of the Transwell plate (i.e., the bottom of the 24-well plate), and add 100-150μL of cell suspension to the upper chamber. Continue incubation for 24-48 hours. Carefully remove the upper chamber with forceps, aspirate the liquid in the upper chamber, and transfer it to wells pre-filled with about 800μL of methanol. Fix at room temperature for 60 minutes. Remove the upper chamber, aspirate the fixative solution in the upper chamber, and transfer it to wells pre-filled with about 800μL of crystal violet staining solution. Stain at room temperature for 15-30 minutes. Gently rinse and soak several times with water, remove the upper chamber, aspirate the liquid in the upper chamber, and carefully wipe away the cells on the membrane surface at the bottom of the upper chamber with a moistened cotton swab. Wash each well with 100μL of PBS 1-3 times each time. Count the cells in 5 random fields under a microscope and statistically analyze the results.

[0046] Experimental results are as follows Figure 3 As shown, the results indicate that inhibiting endogenous SLC10A3 can significantly suppress the migration and invasion of gastric cancer cells.

[0047] Example 4 Small interfering RNA from SLC10A3 promotes ferroptosis in gastric cancer cells. The gastric cancer cell line SGC-7901, treated with small interfering RNA from SLC10A3 in Example 1, was used as a cell model to detect ferroptosis-related indicators, including intracellular total iron levels and reactive oxygen species (ROS) levels. The specific procedures are as follows: Part 1: Colorimetric Detection of Total Iron in Cells I. Preparation 1. Sample preparation Cell samples: Digest and centrifuge the cells in the six-well plate, transfer them to a 1.5ml EP tube, centrifuge, discard the original culture medium, resuspend in 1ml PBS (cell counting can be performed after this step), centrifuge at 3000rpm for 5min; discard PBS, add 0.2ml reagent one, mix well, place on an ice box for lysis for 10min, then centrifuge at 12000rpm for 10min, and collect the supernatant for later use.

[0048] 2. Reagent preparation 1) Before testing, equilibrate the reagents in the kit to room temperature.

[0049] 2) Preparation of 100 μmol / L iron standard: Mix 10 μL of reagent three with 990 μL of double-distilled water until homogeneous. Prepare as needed and use immediately.

[0050] 3) The dilution of standards at different concentrations is shown in Table 1.

[0051] Table 1

[0052] II. Operation 1. Operating Steps 1) Standard wells: Take 40 μL of different concentrations of standard and add them to the corresponding wells of the microplate.

[0053] Measurement wells: Take 40 μL of the sample to be tested and add it to the corresponding well of the ELISA plate.

[0054] 2) Add 40 μL of reagent II to the test well and the standard well.

[0055] 3) Mix well and incubate at 37°C for 40 min.

[0056] 4) Measure the OD value of each well at 593 nm using an ELISA reader.

[0057] 2. The operation table is shown in Table 2.

[0058] Table 2

[0059] III. Result Calculation 1) Fitting curve for standard sample: y = ax + b; Cell sample (by cell count): Fe content (nmol / 10) 6 ) = ( A - b / a) / (N / V)*f; Where y: OD value of standard - OD value of blank (OD value when the concentration of standard is 0), x: concentration of standard, a: slope of standard curve, b: intercept of standard curve. A: Absolute OD value of the sample (OD value of measurement wells - OD value of blank wells), N: Number of cell samples used for lysis / 10 6 V: Amount of reagent added during cell sample processing (mL), f: Dilution factor of the sample before it is added to the detection system.

[0060] Part Two: ROS Detection (1) Cell seeding: Seed cells in a six-well plate and culture for 24 h to allow them to adhere and grow.

[0061] (2) Probe treatment: Remove the cell culture medium and add 1 mL of DHE probe solution diluted to a final concentration of 10 μM with serum-free DMEM medium to each well.

[0062] (3) Incubation and staining: Place the six-well plate in an incubator and incubate at 37°C for 30 min to allow the DHE probe to fully contact the cells and react.

[0063] (4) Washing cells: After incubation, discard the probe solution and gently wash the cells twice with PBS to remove free probes that have not entered the cells.

[0064] (6) Fluorescence detection: The six-well plate was placed under an inverted fluorescence microscope for observation, and fluorescence images were taken and the fluorescence intensity in the cells was recorded.

[0065] Experimental results are as follows Figure 4 As shown, inhibiting endogenous SLC10A3 can significantly increase the total iron and ROS levels in gastric cancer cells.

[0066] In summary, this invention reveals that SLC10A3 small interfering RNA exerts an anti-tumor effect by inhibiting the proliferation, invasion, and migration of gastric cancer cells and promoting ferroptosis. Inhibiting endogenous SLC10A3 in the SGC-7901 gastric cancer cell line can suppress the proliferation, invasion, and migration of gastric cancer cells and promote ferroptosis. Combining SLC10A3 small interfering RNA with appropriate vectors to form drugs, and introducing them into gastric tissue or the body, will have preventive and therapeutic effects against gastric cancer. It is hoped that SLC10A3 small interfering RNA can be combined with chitosan, cholesterol, liposomes, nanoparticles, etc., to form drug molecules for the prevention and treatment of gastric cancer via oral, intravenous, intramuscular, or direct gastric injection.

[0067] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of SLC10A3 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of SLC10A3 as a target in screening candidate drugs for the prevention and / or treatment of gastric cancer, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

3. Application of SLC10A3 expression inhibitors, knockout agents, or silencing agents in the preparation of drugs for the prevention and / or treatment of gastric cancer.

4. A small interfering RNA of SLC10A3, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.6 or SEQ ID NO.

7.

5. A drug for the prevention and / or treatment of gastric cancer, characterized in that, Includes the small interfering RNA as described in claim 4 and a pharmaceutically acceptable vector.

6. The drug according to claim 5, characterized in that, The carrier includes chitosan, cholesterol, liposomes, or nanoparticles.

7. The drug according to claim 5, characterized in that, The drug exerts its therapeutic or preventive effect on gastric cancer by inhibiting the proliferation, invasion and migration of gastric cancer cells, and promoting ferroptosis of gastric cancer cells.

8. Application of reagents for detecting SLC10A3 expression levels in the preparation of products for gastric cancer diagnosis or prognostic assessment.

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