Application of RNASEK gene as a gastric cancer diagnosis and treatment target

Abstract

The application discloses application of RNASEK gene as a gastric cancer diagnosis and treatment target, and belongs to the technical field of biological medicine. The application provides, in one aspect, application of RNASEK gene as a target in preparation of a gastric cancer treatment drug. Experiments prove that silencing of RNASEK gene (such as siRNA) can significantly inhibit gastric cancer cell proliferation, induce cell membrane damage, and inhibit secretion of VEGF, MMP-2 and TGF-beta 1. In another aspect, the application also provides a gastric cancer diagnosis kit based on qPCR technology for detecting the expression level of RNASEK. The application provides a brand-new specific molecular target and an effective biological technology tool for diagnosis and treatment of gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the RNASEK gene as a therapeutic target for gastric cancer. Background Technology

[0002] Gastric cancer is one of the malignant tumors with high incidence and mortality rates in my country and even globally. Its early symptoms are hidden and lack typical manifestations. Most patients are diagnosed at an advanced stage, missing the best intervention time and resulting in a poor overall clinical prognosis.

[0003] Currently, the clinical diagnosis of gastric cancer mainly relies on gastroscopy, pathological biopsy, and imaging examinations. These methods are generally highly invasive, have insufficient sensitivity in early screening, and limited specificity, making it difficult to meet the clinical needs of large-scale early screening and non-invasive auxiliary diagnosis. Therefore, there is an urgent need to discover highly specific and sensitive molecular biomarkers for early screening and auxiliary diagnosis of gastric cancer. Meanwhile, the clinical treatment of gastric cancer faces challenges such as poor targeting of chemotherapy drugs, easy development of drug resistance, and significant systemic adverse reactions, limiting treatment efficacy. Finding specific therapeutic targets for gastric cancer and developing highly targeted drugs with low toxicity has become a key research focus and urgent need in the field of precision diagnosis and treatment of gastric cancer.

[0004] RNASEK (ribonuclease K), a key functional gene involved in the stable maintenance of the V-ATPase complex and the regulation of endosome acidification, has attracted attention in the field of tumor research in recent years. However, existing research has not reported the functional mechanism of RNASEK in the occurrence and development of gastric cancer, nor has it disclosed any related technologies for using RNASEK as a therapeutic target for gastric cancer, developing targeted siRNA agents, and corresponding diagnostic kits. This invention experimentally demonstrates for the first time that targeted silencing of RNASEK can effectively inhibit the proliferation of gastric cancer cells, induce cell membrane damage, and inhibit the secretion of pro-angiogenesis and pro-invasive factors, providing a novel molecular target for the treatment of gastric cancer, and also providing a new tool for the diagnosis of gastric cancer. Summary of the Invention

[0005] This invention aims to provide the application of the RNASEK gene as a therapeutic target for gastric cancer. The mRNA expression level of the gene in gastric cancer tissue is significantly higher than that in adjacent normal gastric tissue. This invention can solve the technical problems of the lack of highly specific and sensitive molecular markers for gastric cancer diagnosis, as well as the insufficient therapeutic targets and poor targeting in current gastric cancer diagnosis. This invention provides new strategies and theoretical basis for the diagnosis and treatment of gastric cancer.

[0006] In a first aspect, the present invention provides the application of RNASEK as a target in the preparation of drugs for treating gastric cancer.

[0007] Optionally, the target is a nucleic acid molecule of the RNASEK gene or a protein encoded by the RNASEK gene.

[0008] Optionally, the mRNA sequence of the RNASEK gene is shown in SEQ ID NO:1.

[0009] Secondly, this invention provides the application of a reagent for silencing the RNASEK gene in the preparation of drugs for treating gastric cancer.

[0010] The reagents provided by this invention significantly inhibit the proliferation of gastric cancer cells, induce cell membrane damage, and inhibit the secretion of VEGF, MMP-2, and TGF-β1 by gastric cancer cells, providing novel molecular targets and experimental evidence for targeted therapy of gastric cancer.

[0011] Optionally, the reagent is a siRNA targeting the RNASEK gene, the sequence of which is shown in SEQ ID NO:2.

[0012] Optionally, the drug has any one or more of the following effects: (1) Inhibits the proliferation of gastric cancer cells; (2) Induces damage to the gastric cancer cell membrane; (3) Inhibits the secretion of vascular endothelial growth factor (VEGF) by gastric cancer cells; (4) Inhibits the secretion of matrix metalloproteinase MMP-2 by gastric cancer cells; (5) Inhibits the secretion of transforming growth factor TGF-β1 by gastric cancer cells.

[0013] Thirdly, the present invention provides a gastric cancer diagnostic kit.

[0014] The kit provided by this invention can rapidly and sensitively detect the mRNA expression level of RNASEK in samples using RT-qPCR technology. It is suitable for early screening and auxiliary diagnosis of gastric cancer, is easy to operate, and provides accurate results, showing good prospects for clinical application.

[0015] Optionally, the kit contains primers for specifically amplifying the RNASEK gene with sequences as shown in SEQ ID NO:3-4, primers for the internal reference gene GAPDH with sequences as shown in SEQ ID NO:5-6, and a real-time PCR reaction mixture.

[0016] Fourthly, the present invention provides a gastric cancer treatment drug comprising a reagent for silencing the RNA SEK gene and a pharmaceutically acceptable vector.

[0017] Optionally, the reagent is siRNA targeting the RNASEK gene.

[0018] Optionally, the vector includes the pGenesil-1 vector. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0020] Figure 1 This refers to the mRNA expression level of RNASEK in gastric cancer tissue and adjacent normal tissue in Example 1 of this invention; Figure 2 This refers to the level of RNASEK mRNA expression in MKN-45 cells after silencing RNASEK in Example 2 of this invention. Figure 3 This describes the effect of silencing RNA SEK on the viability of MKN-45 cells in Example 2 of this invention. Figure 4 This refers to the effect of silencing RNA SEK on LDH release rate in MKN-45 cells in Example 2 of this invention. Figure 5 This is the effect of silencing RNA SEK on the secretory factor levels of MKN-45 cells in Example 2 of the present invention; where figure a represents VEGF level, figure b represents MMP-2 level, and figure c represents TGF-β1 level. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below with reference to the accompanying drawings and through the following embodiments. However, the following embodiments are illustrative and are only used to specifically describe the present invention, and not to limit the present invention.

[0022] This invention provides the application of RNASEK as a target in the preparation of drugs for treating gastric cancer.

[0023] In some embodiments, the target is a nucleic acid molecule of the RNASEK gene or a protein encoded by the RNASEK gene.

[0024] In some embodiments, the mRNA sequence of the RNASEK gene is shown in SEQ ID NO:1.

[0025] This invention also provides the application of reagents for silencing the RNA SEK gene in the preparation of drugs for treating gastric cancer.

[0026] In some embodiments, the reagent is siRNA targeting the RNASEK gene, the sequence of which is shown in SEQ ID NO:2.

[0027] In some embodiments, the drug has one or more of the following effects: (1) Inhibits the proliferation of gastric cancer cells; (2) Induces damage to the gastric cancer cell membrane; (3) Inhibits the secretion of vascular endothelial growth factor (VEGF) by gastric cancer cells; (4) Inhibits the secretion of matrix metalloproteinase MMP-2 by gastric cancer cells; (5) Inhibits the secretion of transforming growth factor TGF-β1 by gastric cancer cells.

[0028] The present invention also provides a gastric cancer diagnostic kit.

[0029] In some embodiments, the kit comprises primers for specifically amplifying the RNASEK gene with sequences as shown in SEQ ID NO:3-4, primers for the internal reference gene GAPDH with sequences as shown in SEQ ID NO:5-6, and a real-time PCR reaction mixture.

[0030] The present invention also provides a gastric cancer treatment drug comprising a reagent for silencing the RNA SEK gene and a pharmaceutically acceptable vector.

[0031] In some embodiments, the reagent is siRNA targeting the RNASEK gene.

[0032] In some embodiments, the vector includes the pGenesil-1 vector.

[0033] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0034] The present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments. All equivalent transformations made based on the technical solutions of the present invention shall fall within the scope of protection of the present invention.

[0035] 1. Experimental Materials Gastric cancer tissue and adjacent normal gastric tissue samples from 20 patients with gastric cancer were collected. All samples were pathologically confirmed as gastric cancer, and all patients signed informed consent forms. RNASEK primers (SEQ ID NO:3-4) and GAPDH primers (SEQ ID NO:5-6) were synthesized by Shanghai Sangon Biotech Co., Ltd.; TRIzol reagent was purchased from Thermo Fisher Scientific (15596018CN); RNA reverse transcription kit was purchased from Promega Corporation (LS2052); qPCR Master Mix was purchased from Promega Corporation (A6220).

[0036] 2. Experimental Methods (1) Total RNA extraction: Take an appropriate amount of gastric cancer tissue and adjacent normal tissue samples, add TRIzol reagent, and extract total RNA from the samples according to the reagent instructions. Use Nanodrop detector to detect RNA concentration and purity, and select samples with RNA purity (A260 / A280) between 1.8 and 2.0 for subsequent experiments.

[0037] (2) Reverse transcription reaction: Take 1 μg of total RNA and perform reverse transcription reaction to synthesize cDNA according to the reverse transcription kit instructions. Reaction conditions: incubate at 42℃ for 15 min, heat at 85℃ for 5 s to terminate the reaction. Store the obtained cDNA product at -20℃ for later use.

[0038] (3) qPCR detection: Using cDNA as a template, qPCR primers and qPCR Master Mix were added, and the amplification reaction was performed on a real-time quantitative PCR instrument. The reaction conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 40 cycles. GAPDH was used as an internal reference gene, and 2 -ΔΔCt The relative expression level of RNASEK was calculated using this method.

[0039] 3. Experimental Results qPCR results as follows Figure 1 As shown, the relative expression level of RNASEK gene mRNA in gastric cancer tissue was 3.79 times that in adjacent normal gastric tissue (****P < 0.0001), indicating that RNASEK gene is significantly highly expressed in gastric cancer tissue and can serve as a potential specific biomarker for gastric cancer diagnosis.

[0040] 1. Experimental materials: Normal human gastric mucosa GES-1 cells and human gastric cancer MKN-45 cells were purchased from Pronosun Biotechnology Co., Ltd.; RPMI 1640 medium was purchased from Pronosun Biotechnology Co., Ltd. (PM150110); siRNA for silencing RNA SEK (si-RNASEK) and negative control siRNA (si-NC) were purchased from General Biotechnology Co., Ltd.; CCK-8 reagent was purchased from Beyotime Biotechnology Co., Ltd. (C0039); lactate dehydrogenase cytotoxicity assay kit was purchased from Beyotime Biotechnology Co., Ltd. (C0017); human vascular endothelial growth factor assay kit (SEKH-0052), human matrix metalloproteinase 2 assay kit (SEKH-0253), and human transforming growth factor β1 assay kit (SEKH-0316) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; phosphate-buffered saline (PBS) was purchased from Liji Biotechnology Co., Ltd. (AC08L011); qPCR related reagents were the same as in Example 1.

[0041] 2. The experimental reagents were prepared as follows: (1) Cell culture medium: containing 10% fetal bovine serum and 1% streptomycin / penicillin, the remainder being basal culture medium; (2) Phosphate buffer: Dissolve 0.818 g NaCl, 0.037 g KCl, 0.022 g CaCl2, 0.238 g HEPES, and 0.18 g glucose in 90 mL of ultrapure water, adjust the pH to 7.4, and bring the volume to 100 mL for later use; (3) Cell digestion solution: 0.25 g trypsin, add 0.02 g EDTA, add 70 mL PBS solution to dissolve, adjust pH to 7.2-7.4, add ultrapure water to 100 mL, filter to sterilize, and use in cell culture passage; (4) Preparation of CCK-8 working solution: Prepare CCK-8 solution by seeding 100 μL of cell suspension per well in a 96-well plate. The preparation method is as follows: add 10 μL of CCK-8 solution and 90 μL of serum-free culture medium to each well. The CCK-8 solution should be preheated to room temperature before use and should be prepared fresh each time.

[0042] 3. Cellular experimental methods: (1) Cell thawing: Quickly remove the frozen cells from liquid nitrogen and place them in an ice box. Then, quickly thaw the cryovials in a water bath preheated to 37°C and immediately transfer them to a clean bench. Transfer the cell cryopreservation solution to a centrifuge tube containing culture medium and mix thoroughly by pipetting. Centrifuge at 800 rpm and 4°C for 5 min. After centrifugation, discard the supernatant, add 5 mL of culture medium to the cell pellet in the centrifuge tube, and repeatedly pipet to prepare a cell suspension. Transfer the suspension to a culture flask and incubate in a cell culture incubator at 37°C and 5% CO2.

[0043] (2) Cell transfection: Cells can be transfected after 2-3 passages. Before transfection, the cells are seeded in 6-well plates. After the cell growth density reaches 40%-50% confluence and the cells are observed to be in good condition, transfection can be performed.

[0044] (3) RT-qPCR was used to verify the knockdown effect of the RNASEK cell line, using the same method as in Example 1.

[0045] (4) Cell viability assay: At 0 h, 24 h, 48 h, and 72 h post-transfection, 10 μL of CCK8 reagent was added to each well. After incubation in an incubator for 1 h, the absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula: .

[0046] (5) LDH release rate detection: 48 h after transfection, add an appropriate amount of LDH release reagent to the control wells (cell wells without drug treatment for subsequent lysis), pipette and continue incubation. After the predetermined time, centrifuge at 400×g for 5 min, take the supernatant and add it to the detection working solution, incubate at room temperature for 30 min, and then measure the absorbance at 490 nm using a microplate reader to calculate the LDH release rate. The calculation formula is: .

[0047] (6) Detection of secreted factors: Cell culture supernatant was collected 48 h after transfection and centrifuged at 3000 rpm for 10 min to remove cell debris. The human VEGF, MMP-2, and TGF-β1 ELISA kits were used. The corresponding detection reagents were added, and after incubation, the absorbance was measured at the wavelength specified in the instructions using an ELISA reader. The concentrations of VEGF, MMP-2, and TGF-β1 in each sample were calculated according to the formula provided in the kit.

[0048] Experimental results: (1) qPCR results are as follows Figure 2 As shown, compared with the si-NC negative control group, the relative expression level of RNASEK gene in MKN-45 cells transfected with si-RNASEK decreased by 49.7% (***P < 0.001).

[0049] (2) The CCK8 results are as follows Figure 3 As shown, compared with the si-NC negative control group, the viability of MKN-45 cells transfected with si-RNASEK decreased by 15.4% (*P < 0.05), 36.2% (****P < 0.0001), and 18.6% (****P < 0.0001) at 24 h, 48 h, and 72 h after transfection, respectively, indicating that si-RNASEK can significantly inhibit the proliferation of MKN-45 cells, and the inhibitory effect is time-dependent.

[0050] (3) LDH release rate results are as follows Figure 4 As shown, compared with the si-NC negative control group, the LDH release rate of the si-RNASEK transfection group increased by 182% (**P < 0.01), indicating that silencing RNASEK can significantly induce gastric cancer cell membrane damage.

[0051] (4) ELISA test results are as follows Figure 5As shown, compared with the si-NC negative control group, the secretion levels of VEGF, MMP-2 and TGF-β1 in the culture supernatant of MKN-45 cells transfected with si-RNASEK decreased by 38.8% (*P < 0.05), 29.9% (**P < 0.01) and 48.2% (**P < 0.01), respectively, indicating that silencing RNASEK can significantly inhibit the secretion of pro-angiogenic, invasion and metastasis and immune escape-related factors in gastric cancer cells.

[0052] All experimental data in this embodiment of the invention are expressed as mean ± standard error. Statistical analysis was performed using GraphPad Prism 8.0 software. Independent samples t-test was used to compare two groups, and P < 0.05 was considered statistically significant.

[0053] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. The application of RNASEK as a target in the preparation of drugs for treating gastric cancer, characterized in that, The target is a nucleic acid molecule of the RNASEK gene or a protein encoded by the RNASEK gene, and the mRNA sequence of the RNASEK gene is shown in SEQ ID NO:

1.

2. The application of a reagent for silencing the RNA SEK gene in the preparation of drugs for treating gastric cancer, characterized in that... The reagent contains siRNA targeting the RNASEK gene, as shown in SEQ ID NO:

2.

3. The application according to claim 2, characterized in that, The drug has one or more of the following effects: (1) Inhibits the proliferation of gastric cancer cells; (2) Induces damage to the gastric cancer cell membrane; (3) Inhibits the secretion of vascular endothelial growth factor (VEGF) by gastric cancer cells; (4) Inhibits the secretion of matrix metalloproteinase MMP-2 by gastric cancer cells; (5) Inhibits the secretion of transforming growth factor TGF-β1 by gastric cancer cells.

4. A gastric cancer diagnostic kit, characterized in that, Include: (1) Primers for specific amplification of the RNASEK gene with sequences as shown in SEQ ID NO:3-4; (2) Primers for the internal reference gene GAPDH with sequences as shown in SEQ ID NO:5-6; (3) Real-time PCR reaction mixture.

5. A drug for treating gastric cancer, characterized in that, Reagents containing the silenced RNA SEK gene and pharmaceutically acceptable vectors.

6. The drug according to claim 5, characterized in that, The reagent is a siRNA targeting the RNASEK gene, and the vector includes the pGenesil-1 vector.

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