SiRNA for targeted inhibition of DEP-1 gene expression and application thereof

Abstract

The invention belongs to the field of new application of drugs, and particularly relates to siRNA for targeted inhibition of DEP-1 gene expression and application of the siRNA. The invention finds that the proliferation activity and migration ability of cancer cells can be significantly inhibited by specifically inhibiting the expression of DEP-1 gene in gastric cancer cells. Based on the discovery, molecular tools such as DEP-1 targeting small interfering RNA (siRNA) and the like are further provided as a core strategy for developing novel gastric cancer treatment drugs. The achievement provides key evidence for deep understanding of biological functions of DEP-1 in occurrence and development of gastric cancer, also establishes a novel treatment target with potential, points out a new research and development direction for development of gastric cancer targeted drugs with high selectivity and low toxic and side effects, and has important conversion medical value.

Description

Technical Field

[0001] This invention belongs to the field of novel uses of pharmaceuticals, and more specifically, relates to an siRNA that targets and inhibits the expression of the DEP-1 gene and its application. Background Technology

[0002] Gastric cancer is the fifth leading cause of cancer incidence worldwide. Due to the lack of effective early diagnostic biomarkers, gastric cancer patients are often diagnosed at an advanced stage, a major reason for its 5-year survival rate of less than 30%. Existing targeted therapies (such as anti-HER2 therapy) are only effective for some patients and are prone to drug resistance. Nucleic acid drugs, with their programmable sequences, can precisely target specific genes or proteins, effectively circumventing drug resistance while reducing damage to normal cells. Therefore, the discovery of novel therapeutic targets for gastric cancer has become the core technical challenge of this invention. Summary of the Invention

[0003] The purpose of this invention is to provide an siRNA that targets and inhibits the expression of the DEP-1 gene and its application, so as to solve the above-mentioned technical problems.

[0004] This invention provides a siRNA that targets and inhibits the expression of the DEP-1 gene. The siRNA is selected from one of siDEP-1-1 and siDEP-1-2. The sense strand sequence of siDEP-1-1 is shown in SEQ ID NO.1, and the antisense strand sequence is shown in SEQ ID NO.2. The sense strand sequence of siDEP-1-2 is shown in SEQ ID NO.3, and the antisense strand sequence is shown in SEQ ID NO.4.

[0005] The role of the DEP-1 gene in tumors remains unclear; some studies suggest it may inhibit tumor growth, while others indicate it promotes tumor progression. To clarify the specific function of DEP-1 in gastric cancer, this invention constructed DEP-1-targeting siRNAs (siDEP-1-1 and siDEP-1-2) and performed gene knockdown experiments in the human gastric cancer cell line SGC-7901. Simultaneously, DEP-1 expression was enhanced in HGC-27 cells using the overexpression plasmid pcDNA-DEP-1. Experimental results showed that DEP-1 knockdown significantly inhibited gastric cancer cell proliferation and migration and affected the expression of epithelial-mesenchymal transition (EMT)-related proteins; while DEP-1 overexpression produced the opposite effect. These findings systematically reveal the pro-cancer role of DEP-1 in gastric cancer, thus establishing the idea of ​​inhibiting DEP-1 expression as a novel therapeutic approach for gastric cancer.

[0006] The present invention also provides the application of the siRNA in the preparation of drugs for treating gastric cancer.

[0007] Furthermore, the siRNA is used to inhibit the proliferation and migration of gastric cancer cells, as well as to inhibit the expression of E-cadherin and vimentin in gastric cancer cells.

[0008] The present invention also provides a gastric cancer treatment drug, wherein the drug uses the above-mentioned siRNA as the active ingredient.

[0009] Furthermore, the content of siRNA in the drug is 0.1wt%~99wt%.

[0010] Furthermore, the drug comprises siRNA and pharmaceutically acceptable excipients.

[0011] Furthermore, the excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0012] Furthermore, the drug is administered orally or by injection.

[0013] Furthermore, the drug is in the form of a solution, which is a solution composed of water and the siRNA, or a solution composed of physiological saline and the siRNA.

[0014] Beneficial effects: This invention demonstrates through cell experiments that inhibiting DEP-1 expression effectively suppresses the proliferation, colony formation, migration, and invasion of gastric cancer cells, and regulates the expression of EMT markers such as E-cadherin and vimentin, thereby halting gastric cancer progression. This discovery not only clarifies the oncogenic role of DEP-1 in gastric cancer but also provides a promising prospect for the application of highly specific inhibitors, such as siRNA, as targeted drugs for gastric cancer. Compared to traditional chemotherapy, the DEP-1-targeted therapy strategy has advantages such as a clear mechanism of action, high selectivity, and potentially lower side effects, laying a solid experimental foundation for the development of novel gastric cancer treatments and possessing significant clinical translational value. Attached Figure Description

[0015] Figure 1 The graph shows that siRNA targeting DEP-1 reduced the mRNA and protein levels of DEP-1 in SGC-7901 cells. In the graph, A is the statistical graph of the relative expression level of DEP-1 mRNA, B is the immunoblot map of DEP-1 expressed protein, and C is the immunoblot statistical analysis graph.

[0016] Figure 2 The figures show the results of CCK-8 and colony formation experiments on SGC-7901 cells transfected with siDEP-1-1. In the figure, A is a statistical graph of the CCK-8 experiment results, and B is a graph of the colony formation experiment results.

[0017] Figure 3Transwell assay of SGC-7901 cells transfected with siDEP-1-1.

[0018] Figure 4 Figure showing the scratch healing experiment of SGC-7901 cells transfected with siDEP-1-1.

[0019] Figure 5 The figure shows the effect of siDEP-1-1 on the expression of E-cadherin and vimentin in SGC-7901 cells detected by Western blot. A is the immunoblot map of the protein, and B is the immunoblot statistical analysis graph.

[0020] Figure 6 The graph shows the level of DEP-1 in HGC-27 cells after transfection with pcDNA3.1 and pcDNA-DEP-1. In the graph, A is the statistical graph of the relative expression level of DEP-1 mRNA, B is the immunoblot map of DEP-1 expressed protein, and C is the immunoblot statistical analysis graph.

[0021] Figure 7 Statistical graph of the results of CCK-8 experiments on HGC-27 cells transfected with pcDNA-DEP-1.

[0022] Figure 8 The image shows the clonogenic capacity of HGC-27 cells transfected with pcDNA-DEP-1, where A is the clonogenic capacity graph and B is the clonogenic capacity statistical graph.

[0023] Figure 9 This is a Transwell experiment diagram of HGC-27 cells transfected with pcDNA-DEP-1.

[0024] Figure 10 This image shows the scratch healing experiment of HGC-27 cells transfected with pcDNA-DEP-1.

[0025] Figure 11 The figure shows the effect of pcDNA-DEP-1 on the expression of E-cadherin and vimentin in HGC-27 cells as detected by Western blot. In the figure, A is the immunoblot map of E-cadherin and vimentin, and B is the immunoblot statistical graph. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0027] Example 1 I. Experimental Materials.

[0028] 1. Cell lines and cell culture: Two human gastric cancer cell lines, SGC-7901 and HGC-27, were purchased from the China Type Culture Collection Committee (Shanghai, China). SGC-7901 and HGC-27 were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (WISENT), 100 U / mL penicillin, and 100 mg / mL streptomycin. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0029] 2. Nucleotide transfection: siDEP-1 and its negative control were synthesized by Gemma Gene (Shanghai, China). The overexpression plasmid pcDNA3.1 was purchased from Ubisoft Bio. SGC-7901 cells were transfected with oligonucleotides siDEP-1-1, siDEP-1-2, and siNC using RFect siRNA / miRNA transfection reagent (catalog number: 11013, Baidai Biotechnology, Changzhou, China) according to the manufacturer's protocol, while HGC-27 cells were transfected with pcDNA3.1 and pcDNA-DEP-1. The siRNA sequences used in the experiments are shown in Table 1.

[0030] Table 1: siRNA sequences 3. RNA Extraction and qRT-PCR: Total RNA was extracted from gastric cancer cell lines using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. cDNA was then synthesized using the FastKing RT kit (containing gDNase) (Tiangen Biotech, China). qRT-PCR was performed on a Quant Studio5 real-time PCR system (Applied Biosystems, USA) using ChamQ Universal SYBR qPCR premix (Vazyme, Nanjing, China).

[0031] II. Experimental Methods.

[0032] 1. Plasmid construction: The cDNA of DEP-1 was inserted into the pcDNA3.1 vector. EcoR I and Xhol Between the restriction enzyme sites. The nucleotide sequence of the cDNA of DEP-1 is shown in SEQ ID NO.7.

[0033]

[0034] 2. Protein Extraction and Western Blotting: Cell pellet was collected and resuspended using lysis buffer. The resuspended cell pellet was then incubated on ice for 20 min, followed by centrifugation at 12000g for 20 min at 4°C. The supernatant was collected and protein concentration was measured using a BCA protein assay kit (Beyotime, Shanghai, China). Lysates were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with TBST in 5% skim milk at room temperature for 2 h and incubated overnight with primary antibody in TBST at 4°C. Subsequently, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 2 h. Protein bands were detected using a BeyoECL Star chemiluminescence assay kit (Beyotime, Shanghai, China) and visualized using a multifunctional protein imaging system (Cell Biosciences, USA). The following antibodies were used: β-actin (protein-tech, 60008-1-Ig), α-tubulin (CST, #3873), GAPDH (protein-tech, 60004-1-Ig), DEP-1 (R&D Systems, AF1934), E-cadherin (CST, #3195), and vimentin (Santa Cruz, sc-6260).

[0035] 3. Cell proliferation assay: Cell proliferation was measured using a Cell Counting Kit-8 (Beyotime, China). Cells (1500 cells / well) were seeded into each well of a 96-well plate and cultured for 5 days. CCK-8 absorbance was measured at 0, 24, 48, 72, and 96 hours after cell culture. 10 μl of CCK-8 solution was added to each well, and absorbance (450 nm) was measured using a SpectraMax iD3 multimicroplate reader (Molecular Devices, USA). Each group had four replicates.

[0036] 4. Colony formation assay: Transfected cells were seeded into six-well plates (800 cells / well) and incubated at 37°C and 5% CO2 for approximately 2 weeks. Proliferating colonies formed from surviving cells were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet solution for 15 minutes, and then counted. A colony with ≥50 cells / clone was defined as a countable colony.

[0037] 5. Scratch healing assay: Transfected cells were seeded in 12-well plates (2 × 10⁻⁶ cells / well). 5 (cell / well). After 24 hours of incubation, scratches were made using a 200 μL pipette tip, cells were washed with PBS, and serum-free culture medium was added. Images were acquired under an inverted microscope at 0 and 48 hours. The experiment was repeated three times.

[0038] 6. Transwell migration assay: Transfected cells were seeded in the upper chamber of serum-free medium. Medium containing 10% FBS (volume fraction) was placed in the lower chamber as a stimulus. After incubation at 37°C for 24 hours, cells were wiped off the upper membrane surface with a cotton swab. Cells were fixed and stained with 0.05% crystal violet solution. Six random fields of view in each chamber were photographed using an inverted microscope.

[0039] 7. Statistical Analysis: Results are expressed as mean ± standard deviation. Statistical differences between groups were analyzed using t-tests with GraphPad Prism 9.5 software. All experiments were repeated at least three times.

[0040] III. Experimental Results.

[0041] To elucidate the role of DEP-1 in gastric cancer cells, CCK-8 assays, clonogenic assays, Transwell assays, and scratch healing assays were performed to detect the proliferation and migration of gastric cancer cells. Figure 1 As shown, targeting DEP-1 with siRNA reduced the mRNA and protein levels of DEP-1 in SGC-7901 cells. siDEP-1-1, which has a high knockdown efficiency, was selected for subsequent experiments. Figure 2 As shown in the results of CCK-8 and colony formation assays, si-DEP-1-1 significantly inhibited the proliferation of SGC-7901 cells. Furthermore, Figure 3 and Figure 4 This indicates that si-DEP-1-1 significantly inhibited cell migration. Finally, as... Figure 5 As shown, knocking down DEP-1 gene expression upregulated E-cadherin expression while downregulated vimentin expression.

[0042] Instead, the pcDNA3.1-DEP-1 plasmid was constructed and transfected into HGC-27 cells. Figure 6 As shown, DEP-1 overexpression was confirmed by qRT-PCR and Western blotting. This contrasts with the results for siDEP-1-1, as... Figure 7 , Figure 8 , Figure 9 , Figure 10 and Figure 11 As shown, the CCK-8 assay, colony formation assay, Transwell assay, scratch healing assay, and E-cadherin and vimentin analysis of pcDNA-DEP-1 transfected cells all showed contradictory results. In summary, these results suggest that directly targeting and silencing DEP-1 can inhibit the proliferation, migration, and expression of E-cadherin and vimentin in gastric cancer cells.

[0043] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0044] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0045] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A siRNA that targets and inhibits the expression of the DEP-1 gene, characterized in that, The siRNA is selected from one of siDEP-1-1 and siDEP-1-2. The sense strand sequence of siDEP-1-1 is shown in SEQ ID NO.1, and the antisense strand sequence is shown in SEQ ID NO.

2. The sense strand sequence of siDEP-1-2 is shown in SEQ ID NO.3, and the antisense strand sequence is shown in SEQ ID NO.

4.

2. The use of the siRNA according to claim 1 in the preparation of a gastric cancer treatment drug.

3. The application of siRNA according to claim 2 in the preparation of a gastric cancer therapeutic drug, characterized in that, The siRNA is used to inhibit the proliferation and migration of gastric cancer cells, as well as to inhibit the expression of E-cadherin and vimentin in gastric cancer cells.

4. A drug for treating gastric cancer, characterized in that, The drug uses the siRNA described in claim 1 as its active ingredient.

5. The drug according to claim 4, characterized in that, The content of siRNA in the drug is 0.1wt%~99wt%.

6. The drug according to claim 4, characterized in that, The drug includes siRNA and pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

8. The drug according to claim 4, characterized in that, The drug can be administered orally or by injection.

9. The drug according to claim 4, characterized in that, The drug is in the form of a solution, which is a solution composed of water and the siRNA, or a solution composed of physiological saline and the siRNA.

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