Application of CDH18 gene as target spot in screening medicine for preventing or treating cervical cancer

Abstract

The invention relates to the technical field of biology, in particular to application of a CDH18 gene serving as a target spot to screening of drugs for preventing or treating cervical cancer, the drugs take the CDH18 gene as the target spot, expression of the CDH18 gene is inhibited or silenced, the proliferation and migration capacity of cervical cancer cells can be enhanced by regulating and controlling a Wnt pathway through overexpression of the CDH18 gene, and the drugs for preventing or treating cervical cancer can be used for preventing or treating cervical cancer. The down-regulation of the CDH18 gene inhibits the expression of beta-catenin, the knock-down of the CDH18 gene can inhibit the proliferation and migration ability of cervical cancer cells by regulating and controlling a Wnt signal pathway, the kaempferide has a certain potential anti-tumor activity effect in the cervical cancer cells, the kaempferide can induce the apoptosis of SiHa and HeLa cells of cervical cancer, and the kaempferide can inhibit the proliferation and migration ability of the cervical cancer cells. Experiments show that the kaempferide inhibits the expression level of Wnt signal pathway related molecules by reducing the expression level of CDH18, further promotes apoptosis of cervical cancer cells and inhibits proliferation and migration of the cervical cancer cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to... CDH18 The application of genes as targets in screening drugs for the prevention or treatment of cervical cancer. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors among women worldwide, seriously threatening women's health and lives. Human papillomavirus (HPV) infection is the most common causative factor. The virus's DNA integrates into the host genome, leading to dysregulation of key genes and malignant transformation of cells, ultimately progressing to invasive carcinoma. Cervical cancer is mainly divided into squamous cell carcinoma and adenocarcinoma. Squamous cell carcinoma is the most common, accounting for approximately 70%-80% of all cervical cancers, originating from the squamous epithelium of the external os of the cervix. Adenocarcinoma accounts for approximately 15%-20%, originating from the glandular epithelium within the cervical canal. Its incidence has shown a relatively upward trend in recent years, possibly related to differences in the detection rate of glandular epithelial lesions in screening methods. Although significant progress has been made in the prevention and early treatment of cervical cancer with the widespread adoption of screening and HPV vaccination, the treatment outcomes for advanced and recurrent cervical cancer remain unsatisfactory, and it is a major cause of death among patients.

[0003] In delving into the molecular mechanisms of cervical cancer to identify new therapeutic targets, current research has moved beyond traditional histopathology and entered an era of precision medicine centered on driver genes, signaling pathways, and the tumor microenvironment. Clearly understood targets or signaling pathways include HPV oncoproteins (E6 / E7), the PI3K / AKT / mTOR pathway, the vascular endothelial growth factor pathway, and the epidermal growth factor receptor pathway. Target research in cervical cancer is progressing from single pathways to network regulation, from tumor cells themselves to the tumor microenvironment, and from traditional targeted therapies to combined immunotherapy strategies. However, many unknown driver molecules and drug resistance mechanisms remain to be elucidated.

[0004] Cadmin 18, also known as CDH14, is an important member of the type II classical cadherin family. It is a cell membrane adhesion molecule primarily expressed in the reproductive and nervous systems and is involved in the occurrence and development of various diseases. Literature reports that CDH18 expression is significantly higher in cancers such as lung squamous cell carcinoma (LUSC), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pheochromocytoma and paraganglioma (PCPG), endometrial cancer of the uterine corpus (UCEC), and invasive breast cancer (BRCA) than in adjacent normal tissues. However, whether cadherin 18 can be used as a target for screening drugs for the treatment of cervical cancer has not been reported in the literature. Summary of the Invention

[0005] The primary objective of this invention is to provide CDH18The application of genes as targets in screening drugs for the prevention or treatment of cervical cancer, wherein the drugs are... CDH18 Targeting genes to suppress or silence them CDH18 Gene expression.

[0006] A second objective of this invention is to provide suppression CDH18 Application of gene expression reagents in the preparation of drugs for the prevention or treatment of cervical cancer.

[0007] A third objective of this invention is to provide suppression CDH18 Application of gene-expressing drugs in the preparation of drugs for the prevention or treatment of cervical cancer.

[0008] Preferably, the inhibition CDH18 Reagents or inhibitors of gene expression CDH18 Drugs for gene expression include one or more of the following: shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies.

[0009] Preferably, the drug is kaempferol.

[0010] The fourth objective of this invention is to provide CDH18 Application of genes in inhibiting the replication of cervical cancer cells.

[0011] The fifth object of the present invention is to provide an anti-cervical cancer pharmaceutical composition, characterized in that the composition comprises CDH18 Inhibitors of genes or proteins, or CDH18 A combination of gene or protein inhibitors and antitumor drugs.

[0012] Preferably, the pharmaceutical composition is characterized in that it further comprises pharmaceutically acceptable excipients.

[0013] The sixth objective of this invention is to provide a CDH18 Gene knockout cell lines, wherein the cell lines are generated by gene editing technology. CDH18 Obtained by gene knockout.

[0014] The seventh objective of this invention is to provide the use of kaempferol in the preparation of drugs for the prevention or treatment of cervical cancer.

[0015] The beneficial effects of the present invention are: (1) The present invention provides a solution for providing CDH18 The application of genes as targets in screening drugs for the prevention or treatment of cervical cancer, wherein the drugs are... CDH18 Targeting genes to suppress or silence them CDH18 Gene expression. Overexpression. CDH18 Genes can enhance the proliferation and migration of cervical cancer cells by regulating the Wnt pathway. CDH18 Downregulation of the gene inhibited β-catenin expression; knockdown... CDH18 Genes can inhibit the proliferation and migration of cervical cancer cells by regulating the Wnt signaling pathway. Overexpression of [the gene / gene / etc.] CDH18 Adding XAV939 to SiHa and HeLa cells significantly eliminated the gene. CDH18 The role of genes in promoting the proliferation and migration of cervical cancer cells.

[0016] (2) MTT results showed that the survival rates of both SiHa and HeLa cells decreased in a concentration-dependent manner, and the IC50 of kaempferol on SiHa and HeLa cells was determined. 50 The concentrations were 15.19 μmol / L and 35.47 μmol / L, respectively. Meanwhile, Western blot results also confirmed that kaempferol could significantly inhibit the activity of kaempferol in cervical cancer cells SiHa and HeLa. CDH18 Protein expression levels. Kaempferol reversed [the effect]. CDH18 Gene activation of the Wnt pathway can mitigate the progression of cervical cancer.

[0017] (3) The results showed that kaempferol has certain potential anti-tumor activity in cervical cancer cells, further clarifying its potential as an anti-cancer drug. Kaempferol can induce apoptosis in cervical cancer SiHa and HeLa cells, and further clarified that kaempferol works by reducing the tumor growth rate of cervical cancer cells. CDH18 Gene expression promotes apoptosis. Transwell assays were used to further investigate the effect of kaempferol on cell membrane penetration, revealing a significant dose-dependent inhibitory effect. Kaempferol also exhibits similar tumor-inhibiting effects in vivo. Kaempferol demonstrates good therapeutic efficacy against tumors in nude mice, significantly slowing tumor growth. In vivo and in vitro experiments show that kaempferol promotes apoptosis in cervical cancer cells and inhibits their proliferation and migration by reducing CDH18 expression levels and thereby inhibiting the expression levels of molecules related to the Wnt signaling pathway. Attached Figure Description

[0018] Figure 1 Overexpression in cervical cancer cells CDH18 Genes promote the expression of β-catenin; Note: (A) STRING database predicts molecules that interact with CDH18 protein; (B) Western blot verifies the transfection efficiency of SiHa and HeLa cells; (C) Western blot detects β-catenin protein expression level; Figure 2 Overexpression in cervical cancer cells CDH18A bar chart showing how gene expression promotes β-catenin expression; Note: (A) Image J analysis of CDH18 protein level differences in SiHa cells; (B) Image J analysis of CDH18 protein level differences in HeLa cells; (C) Image J analysis of β-catenin protein level differences in SiHa cells; (D) Image J analysis of β-catenin protein level differences in HeLa cells; Figure 3 overexpression CDH18 The gene enhances the proliferation and migration of cervical cancer cells through the Wnt signaling pathway; Note: (A) Western blot was used to detect the expression levels of proteins related to the Wnt signaling pathway; (B) EdU assay was used to detect cell proliferation (×100); (C) Transwell assay was used to detect cell migration (×100). Figure 4 overexpression CDH18 Bar chart showing how genes enhance the proliferation and migration of cervical cancer cells through the Wnt signaling pathway; Note: (A) Image J analysis of differences in c-Myc protein levels in SiHa cells; (B) Image J analysis of differences in c-Myc protein levels in HeLa cells; (C) Image J analysis of differences in TCF4 protein levels in SiHa cells; (D) Image J analysis of differences in TCF4 protein levels in HeLa cells; (E) Image J analysis of HeLa cell proliferation capacity (×100); (F) Image J analysis of SiHa cell proliferation capacity (×100); (G) Image J analysis of SiHa cell migration capacity; (H) Image J analysis of HeLa cell migration capacity; (I) Image J analysis of SiHa cell migration capacity; (J) Image J analysis of HeLa cell migration capacity; Figure 5 Wound healing assays detect cell migration ability; Note: The attached diagram is ×100.

[0019] Figure 6 Knockdown in cervical cancer cells CDH18 Genetic repression of β-catenin expression; Note: (A) Western blot to verify the transfection efficiency of SiHa and HeLa cells; (B) Western blot to detect the expression level of β-catenin protein; (C) Western blot to verify the transfection efficiency of SiHa and HeLa cells (bar chart); (D) Western blot to detect the expression level of β-catenin protein (bar chart). Figure 7 Knock down CDH18 The gene inhibits the proliferation and migration of cervical cancer cells through the Wnt signaling pathway; Note: (A) Western blot was used to detect the expression levels of proteins related to the Wnt signaling pathway; (B) EdU assay was used to detect cell proliferation (×100); (C) Transwell assay was used to detect cell migration (×100). Figure 8 Knock down CDH18 Bar chart showing the ability of genes to inhibit the proliferation and migration of cervical cancer cells through the Wnt signaling pathway; Note: (A) Image J analysis of differences in c-Myc protein levels in SiHa cells; (B) Image J analysis of differences in c-Myc protein levels in HeLa cells; (C) Image J analysis of differences in TCF4 protein levels in SiHa cells; (D) Image J analysis of differences in TCF4 protein levels in HeLa cells; (E) Image J analysis of SiHa cell migration ability; (F) Image J analysis of HeLa cell migration ability. Figure 9 Wound healing assays detect cell migration ability; Note: The attached diagram is ×100.

[0020] Figure 10 Wound healing assays were used to assess cell proliferation and migration abilities. A: Image J analysis of SiHa cell proliferation ability; B: Image J analysis of HeLa cell proliferation ability; C: Image J analysis of SiHa cell migration ability; D: Image J analysis of HeLa cell migration ability; Figure 11 XAV939 inhibits the expression of the Wnt signaling pathway; Note: (A) Structural formula of XAV939; (B) Molecular docking binding energy score of XAV939 and β-catenin; (C) Schematic diagram of molecular docking of XAV939 and β-catenin; (D) MTT assay for IC50 of XAV939 against cervical cancer cells. 50Value; (E) Western blot detection of CDH18 protein expression level in cervical cancer cells after adding different concentrations of XAV939; (F) Western blot detection of Wnt pathway-related molecule protein expression level; Figure 12 XAV939 inhibits the expression of the Wnt signaling pathway; Note: (A) Image J analysis of differences in β-catenin protein levels in SiHa cells; (B) Image J analysis of differences in β-catenin protein levels in HeLa cells; (C) Image J analysis of differences in c-Myc protein levels in SiHa cells; (D) Image J analysis of differences in c-Myc protein levels in HeLa cells; (E) Image J analysis of differences in TCF4 protein levels in SiHa cells; (F) Image J analysis of differences in TCF4 protein levels in HeLa cells; Figure 13 XAV939 inhibits the proliferation and migration of cervical cancer cells through the Wnt signaling pathway; Note: (A) EdU assay for cell proliferation (×100); (B) Transwell assay for cell migration (×200); (C) Wound healing assay for cell migration (×100). Figure 14 XAV939 inhibits the proliferation and migration of cervical cancer cells via the Wnt signaling pathway (bar chart); Note: (A) Image J analysis of EdU assay bar chart for cell proliferation capacity; (B) Image J analysis of Transwell assay bar chart for cell migration capacity; (C) Image J analysis of wound healing assay bar chart for cell migration capacity. Figure 15 Bioinformatics analysis of the interaction between CDH18 protein and β-catenin protein; Note: (AB) STRING network diagram shows the correlation between CDH18 and CTNNB1 (β-catenin); (C) Hydrogen bond visualization diagram shows the molecular docking results of CDH18 and β-catenin, with pink representing CDH18 molecules and light blue representing β-catenin molecules; (D) Molecular docking binding energy score of CDH18 and β-catenin. Figure 16 Kaempferol's inhibitory effect on the growth of cervical cancer cells; Note: (A) Structural formula of kaempferol; (B) Molecular docking binding energy score of kaempferol and CDH18; (C) Schematic diagram of molecular docking of kaempferol and CDH18; (D) MTT assay for IC50 of kaempferol against cervical cancer cells.50 Value; (E) Western blot analysis of the effect of different concentrations of kaempferol on CDH18 protein expression level in cervical cancer cells; (F) Image J analysis of the differences in CDH18 protein expression level; Figure 17 Kaempferol inhibits CDH18 Genes regulate the Wnt signaling pathway and modulate the expression levels of related molecular proteins; Figure 18 Kaempferol inhibits CDH18 Bar chart showing gene regulation of the Wnt signaling pathway and the regulation of expression levels of related molecular proteins; Note: (A) Image J analysis of β-catenin protein expression levels in SiHa and HeLa cells; (B) Image J analysis of p-β-catenin protein expression levels in SiHa and HeLa cells; (C) Image J analysis of GSK-3β protein expression levels in SiHa and HeLa cells; (D) Image J analysis of c-Myc protein expression levels in SiHa and HeLa cells; (E) Image J analysis of TCF4 protein expression levels in SiHa and HeLa cells; Figure 19 Kaempferol has the ability to inhibit the proliferation of cervical cancer cells in vitro; Note: (A) Western blot detection in overexpression CDH18 (A) CDK4 protein expression level in SiHa and HeLa cells after the addition of kaempferol; (B) Image J analysis of the differences in CDK4 protein expression level; (C) EdU method to detect the effect of kaempferol on cell proliferation; (D) Image J analysis of the bar chart of cell proliferation capacity detected by EdU method; (E) MTT method to detect the effect of kaempferol on cell viability; Figure 20 Kaempferol promotes apoptosis in cervical cancer cells in vitro; Note: (A) Western blot detection in overexpression CDH18 (A) Expression levels of Bax, Bcl-2, caspase 3, and cleaved caspase 3 proteins in SiHa and HeLa cells after the addition of kaempferol; (B) Image J analysis of differences in protein expression levels; Figure 21 The effect of kaempferol on the number of apoptotic cells was detected by Hoechst 33258 nuclear staining method. Figure 22 Kaempferol inhibits the migration of cervical cancer cells in vitro; Note: (A) Cell scratch assay to detect the effect of kaempferol on cell migration ability; (B) Image J analysis of cell migration ability bar chart; (C) Transwell assay to detect the effect of kaempferol on cell migration ability; (D) Image J analysis of cell migration ability bar chart.

[0021] Figure 23 Effects of kaempferol on tumor growth in nude mice; Note: (A) Establishment of a nude mouse tumor model; (B) Images of xenograft tumors in nude mice in the empty cell group and the drug-treated group; (C) Statistical analysis of the differences in tumor volume between the two groups of nude mice xenograft tumors; (D) Statistical analysis of the differences in body weight between the two groups of nude mice.

[0022] Figure 24 The effect of kaempferol on the expression levels of Wnt signaling pathway molecules and proteins in vivo; Note: (A) Immunohistochemical assay was used to determine the effect of kaempferol on the expression levels of proteins such as CDH18, β-catenin, GSK-3β, c-Myc and TCF4; (B) Image J analysis was used to analyze the differences in protein expression levels. Detailed Implementation

[0023] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations. The following embodiments are further explanations and supplements to the present invention and do not constitute any limitation on the present invention.

[0024] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0025] It should be noted that in the following examples, ImageJ was used to process the data, and the data represent the average of three independent experiments. All statistical analyses were performed using SPSS 26.0, and comparisons between two sets of data were conducted using... t For comparisons between two or more groups of data, a nonparametric rank-sum test is used. P <0.05 indicates a statistically significant difference. *: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001.

[0026] XAV939 is a selective inhibitor of the Wnt / β-catenin signaling pathway. Its main function is to highly selectively inhibit the two enzymes Tankyrase 1 and Tankyrase 2.

[0027] Example 1 CDH18 The role of genes in the proliferation and migration of cervical cancer cells 1. Materials and Methods 1.1 Main Reagents and Instruments SiHa and HeLa cell lines required for cell culture were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; EdUImaging Kits (Cy3) were purchased from Shanghai Weihuan Biotechnology Co., Ltd.; PEI transfection reagent was purchased from Wuhan Sanying Biotechnology Co., Ltd.; XAV939 was purchased from MCE (MedChemExpress). CDH18 Gene overexpression and knockdown plasmids, Shanghai Gemma Pharmaceutical Technology Co., Ltd.; CDH18 polyclonal antibody, Hangzhou Huaan Biotechnology Co., Ltd.; TCF4 monoclonal antibody, Shanghai Ebibio Biotechnology Co., Ltd.; c-Myc monoclonal antibody, Beijing Biosen Biotechnology Co., Ltd.; β-catenin monoclonal antibody, Wuhan Sanying Biotechnology Co., Ltd.; MouseAnti-GAPDH mAb and goat anti-mouse IgG / horseradish peroxidase labeling, Zhongshan Jinqiao.

[0028] 1.2 Cell culture and drug preparation Cells were cultured in a CO2 incubator at 37°C using complete medium supplemented with 10%-15% fetal bovine serum and 500 μL of penicillin and streptomycin. Meanwhile, XAV939 was diluted with DMSO to a stock solution of 20 μmol / L, and then diluted to the appropriate concentration for subsequent experiments.

[0029] 1.3 Construction of stable transfection cell lines SiHa and HeLa cell lines were stably transfected separately. Cells were seeded in 6-well plates to achieve a cell density of approximately 70%. PEI transfection reagent was used for stable transfection. After 48-72 hours, successfully transfected cells were selected (NC knockdown control group and knockdown control group were established). CDH18 Genomics, NC overexpression genome and overexpression CDH18The genomes were named SiHa shNC group, SiHa sh1050 group, SiHa sh2603 group, SiHa NC-C group, SiHa CDH18 group; HeLa shNC group, HeLash1050 group, HeLa sh2603 group, HeLa NC-C group and HeLa CDH18 group, respectively.

[0030] 1.4 EdU cell proliferation experiment Cells were cultured to the desired density and incubated with 10 μM EdU working solution at 37°C and 5% CO2 for approximately 2 hours. After fixation with 4% paraformaldehyde at room temperature for 15 minutes, the cells were washed three times, and permeabilized with 0.5% Triton X-100 for 20 minutes. Click reaction solution was prepared according to the kit instructions, and the cells were incubated at room temperature in the dark for 30 minutes. The cells were then washed, and the nuclei were stained with DAPI for 5 minutes. Finally, the cells were observed and images were acquired using a fluorescence microscope. Cell proliferation activity was assessed by counting the proportion of EdU-positive cells to the total number of cells.

[0031] 1.5 Cell viability assay Cells are typically 5×10³-1×10⁻⁶ per well. 4 Cells were seeded into 96-well plates with 100 μL of culture medium per well, with at least 5 replicates per group. The plates were then transferred to a 37°C, 5% CO2 incubator for adherence. After cell adhesion, culture medium containing different concentrations of XAV939 was added, and the plates were incubated for predetermined times (e.g., 24 h, 48 h, 72 h). 20 μL of MTT reagent was added to each well, and the plates were incubated for another 3 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The 96-well plates were then shaken on a shaker for 15 min, and the absorbance (OD) value of each well was read using a microplate reader set to 490 nm.

[0032] 1.6 Western blot experiment The absorbance values ​​were read using a microplate reader via the BCA method to calculate the protein concentration of the samples. Based on the quantification results, samples were loaded and then subjected to SDS-PAGE gel electrophoresis, followed by membrane transfer, blocking, and incubation with primary antibody overnight at 4°C. The primary antibody was recovered, and the membrane was washed three times with TBST for 10 min each time. Diluted secondary antibody working solution (generally 1:5000–1:10000) was added, and the membrane was incubated at room temperature for 2 h. The secondary antibody was discarded, and the membrane was washed three times with TBST. The membrane was then placed in a chemiluminescence imaging system, and the exposure time was adjusted according to the signal intensity to acquire band images.

[0033] 1.7 Transwell Experiment For cell counting, add 600 μL of culture medium containing 15% FBS to the lower chamber of a 24-well plate. Place the chamber into the 24-well plate with forceps. Add 200 μL of a mixture of cell suspension and complete culture medium to the upper chamber. Incubate for 36 hours. Gently wipe the cells in the upper chamber with a cotton swab. Fix with 4% paraformaldehyde for 30 minutes. Stain with crystal violet for 30 minutes. Wash three times with PBS to remove unbound crystal violet. Observe and count the cells in five fields of view under a microscope.

[0034] 1.8 Wound Healing Experiment In a six-well plate, ensure that the number of cells in each well is 4 × 10⁶. 5 After culturing the cells in a 37℃ 5% CO2 incubator until the cell confluence rate reaches 100%, use a 200μL pipette tip to make a cut parallel or perpendicular to the horizontal line on the back of the 6-well plate, aligned with the cap or ruler. Wash twice with PBS and add fresh culture medium. Take photos at the same time every day to observe cell migration and healing ability.

[0035] 2. Results 2.1 CDH18 and β-catenin are correlated in cervical cancer cells. A comprehensive analysis of molecules interacting with CDH18 was performed using bioinformatics STRING, and six related genes were identified through core cross-plot screening. Figure 1 A), among which CTNNB1 (β-catenin) showed the highest correlation. Meanwhile, β-catenin is a key molecule in the Wnt signaling pathway, suggesting that CDH18 may regulate the biological function of cervical cancer cells by affecting β-catenin's participation in the Wnt signaling pathway. Secondly, overexpression... CDH18 The gene plasmid was stably transfected into both SiHa and HeLa cell lines, and the successful transfection was verified by Western blot. Figure 1 B, Figure 2 A-2B), the difference was statistically significant. P <0.001). Simultaneously, the expression level of β-catenin protein was detected, and the study found that overexpression... CDH18 Following gene sequencing, the level of β-catenin protein also increases. Figure 1 C, Figure 2 C-2D), the difference was statistically significant ( P <0.01, P <0.001).

[0036] 2.2 Overexpression CDH18 Genes promote the proliferation and migration of cervical cancer cells through the Wnt pathway. The results showed that CDH18Upregulation of the gene activated the expression of c-Myc and TCF4. Figure 3 A, Figure 4 A-4D), the difference was statistically significant ( P <0.001). Overexpression was detected using the EdU assay, Transwell assay, and wound healing assay. CDH18 When the gene was applied, the number of cells proliferated significantly increased compared to the control NC-C group, and the cell membrane penetration and healing abilities were also significantly enhanced. Figure 3 B-3C, Figure 4 E-4J, Figure 5 The difference was statistically significant. P <0.5, P <0.01). In summary, overexpression CDH18 Genes can enhance the proliferation and migration of cervical cancer cells by regulating the Wnt pathway.

[0037] 2.3 Knockdown in cervical cancer cells CDH18 Post-gene repression of β-catenin expression Will knock down CDH18 The gene plasmids were stably transfected into both SiHa and HeLa cell lines, and the protein expression levels of CDH18 and β-catenin were analyzed by Western blot. Figure 6 (A, 6C) The results showed that, compared with the control group, the downregulation of CDH18 inhibited the expression of β-catenin ( Figure 6 B, 6D), the difference was statistically significant ( P <0.5, P <0.001).

[0038] 2.4 Knock Down CDH18 The gene inhibits the proliferation and migration of cervical cancer cells through the Wnt signaling pathway. Knockdown in cervical cancer cells SiHa and HeLa CDH18 Genetic analysis revealed a significant decrease in the expression levels of the proteins c-Myc and TCF4. Figure 7 A, Figure 8 A-8D), the difference was statistically significant ( P <0.01; P <0.001). Knockdown was detected using the EdU assay, Transwell assay, and wound healing assay. CDH18 The study investigated the effects of genes on the proliferation and migration of cervical cancer cells. Results showed that the number of proliferating cells was significantly reduced compared to the control NC group, and the cells' ability to penetrate membranes and heal was also significantly weakened. Figure 7 B-7C, Figure 9 , Figure 8 E-8F, Figure 10 A-10D), the difference was statistically significant ( P <0.5, P <0.01; P <0.001). The results show that knocking down CDH18 Genes can inhibit the proliferation and migration of cervical cancer cells by regulating the Wnt signaling pathway.

[0039] 2.5 XAV939 inhibits the expression of Wnt signaling pathway-related molecules in a dose-dependent manner. In order to evaluate the β-catenin inhibitor XAV-939 ( Figure 11 A) Regarding the effect on Wnt signaling pathway expression, we analyzed the affinity between the two. According to the literature, a docking score of less than -5.0 and greater than -7.0 indicates that the small molecule of the compound has a good binding ability to the target. Figure 11 B). Molecular docking of XAV-939 and β-catenin was performed using the bioinformatics software ZDOCK. Figure 11 C). XAV-939 was added to SiHa and HeLa cells, and several concentration gradients of 1 μmol / L, 2 μmol / L, 4 μmol / L, 8 μmol / L, and 16 μmol / L were set to evaluate the cytotoxic effect of XAV-939 on cells. MTT results showed that the survival rate of both SiHa and HeLa cells decreased in a concentration-dependent manner, and the IC50 values ​​of XAV-939 on SiHa and HeLa cells were 1.719 μmol / L and 1.304 μmol / L, respectively. Figure 11 D). Western blot results also confirmed that XAV-939 inhibited β-catenin protein expression in a dose-dependent manner. Figure 11 E, Figure 12 A-12B), the difference was statistically significant ( P <0.001), while overexpression CDH18 Adding XAV939 to SiHa and HeLa cells significantly reduced the expression of c-Myc and TCF4. Figure 11 F, Figure 12 C-12F), the difference was statistically significant ( P <0.5, P <0.01; P The result of <0.001 further clarifies that CDH18 can affect the molecular expression of the Wnt signaling pathway.

[0040] 2.6 XAV939 inhibits the proliferation and migration of cervical cancer cells through the Wnt signaling pathway. To further investigate whether XAV939 can affect the proliferation and migration of cervical cancer cells by inhibiting the Wnt signaling pathway, XAV939 was overexpressed... CDH18 Adding XAV939 to SiHa and HeLa cells significantly eliminated the gene. CDH18 The promoting effect of genes on the proliferation and migration of cervical cancer cells ( Figure 13 A-13C, Figure 14 A-14C), the difference was statistically significant ( P <0.5, P <0.01; P The result was <0.001, indicating that CDH18 affects the proliferation and migration of cervical cancer cells through the Wnt signaling pathway.

[0041] Example 2: Kaempferol by action CDH18 Genes inhibit the proliferation and migration of cervical cancer cells and promote apoptosis. 1. Materials and Methods 1.1 Chemical reagents and instruments SiHa, HeLa, and HaCaT cell lines required for cell culture were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; CaSki cell line was purchased from Haixing Biotechnology Co., Ltd.; penicillin-streptomycin mixture and trypsin-EDTA digestion solution (phenol red and EDTA-free); RIPA protein lysis buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd.; high glucose medium and fetal bovine serum were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; kaempferol was purchased from MCE; Hoechst apoptosis-nuclear cell kit and BCA protein concentration assay kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; EdU kit was purchased from APE×BIO, USA; primary antibodies: β-catenin (51067-2-AP); CDK4 (66950-1-Ig); cleaved caspase 3 (25128-1-AP); Bcl-2 (12789-1-Ap); caspase 3 (ET1602-39) and Bax (50599-2-Ig) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; CDH18 (ER62866, China) was purchased from Hangzhou Huaan Biotechnology Co., Ltd.; c-Myc (bs-4963R) was purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; TCF4 (CY8810); GSK-3β (CY2434) and p-β-catenin (CY9969) were purchased from Shanghai Ebibio Biotechnology Co., Ltd.; mouse GAPDH monoclonal antibody and goat anti-mouse IgG / horseradish peroxidase label were purchased from Zhongshan Jinqiao.

[0042] 1.2 Cell lines and culture Cells were cultured in a CO2 incubator at 37°C using a complete culture medium containing 10%-15% fetal bovine serum and 500 μL of penicillin and streptomycin. Meanwhile, kaempferol was diluted with DMSO to a stock solution of 20 μmol / L, and then diluted with the stock solution to the appropriate concentration for subsequent experiments.

[0043] 1.3 MTT Experiment Cells were counted and seeded at appropriate densities in five 96-well plates with time gradients (0h, 24h, 48h, 72h, and 96h). Five replicates were set up for each cell group, with 2000 cells seeded in each well. The next day, 20 μL of MTT solution was added in the dark. After incubation for 3 hours, the culture medium was removed, and 150 μL of DMSO solution was added. After shaking for 15 minutes, the absorbance (OD value) of each well was measured at 490 nm using a microplate reader to assess cell viability and growth.

[0044] 1.4 EdU cell proliferation experiment Prepare 96-well plates, seed each well with 100 μL of cell suspension, and incubate overnight at 37 ℃ until cell attachment. Dilute EdU to 20 µM with complete culture medium, add 100 µL of the diluted EdU working solution to each well of the 96-well plate, and incubate at 37 ℃ for 2 hours. Add 50 μL of 4% paraformaldehyde to fix the cells at room temperature for 30 minutes, then wash three times with PBS. Add 100 µL of permeabilization buffer (0.5% Triton X-100 in PBS) to each well and incubate at room temperature for 10-15 minutes, washing three times. Add 100 µL of 1×Hoechst 33342 to each well and stain at room temperature in the dark for 10 minutes, washing three times with PBS. Finally, observe the cells under a fluorescence microscope, photograph and count them.

[0045] 1.5 Immunoprecipitation assay Remove cells with 90% adherence from the large dish, add 1 mL of IP lysis buffer to ice, add an appropriate amount of primary antibody to the protein lysis buffer, and incubate overnight at 4 °C with inversion. The next day, add 30 μL of 50% Protein A / G magnetic beads, incubate for 3 hours, wash the magnetic beads 4-6 times, remove the washing buffer on the last wash, add 2× loading buffer to the precipitate, boil in boiling water for 15 minutes, and detect whether there is an interaction between the two proteins.

[0046] 1.6 Apoptosis – Hoechst Nuclear Staining Assay Prepare 50%-80% adherent cells in a 96-well plate. Fix with fixative for 10 minutes, then wash three times with PBS for 5 minutes each time. Add 200 μL of Hoechst 33258 staining solution from the Hoechst Nuclear Staining Kit, stain for 5 minutes, then wash three times with PBS for 5 minutes each time. Finally, observe the number of apoptotic cells under a fluorescence microscope, take photos, and count them.

[0047] 1.7 Western blot experiment Prepare separating and stacking gels of appropriate concentrations according to molecular weight, load the samples, perform electrophoresis, electrotransfer, transfer to a PVDF membrane, and block in blocking buffer for two hours. After completion, incubate with primary antibody for 12-16 hours, wash three times with 1×TBST for 10 minutes each time, and then incubate with secondary antibody. After incubation, repeat the same steps three times, and then expose and photograph the images for storage.

[0048] 1.8 Wound Healing Experiment Cell counting: Add 2 mL of complete culture medium and cell suspension to each well of a six-well plate to ensure that the number of cells in each well is 400,000. Incubate at 37°C and 5% CO2 for 24 h to achieve 100% cell confluence. On the second day, use a pipette tip to make a cut parallel or perpendicular to the horizontal line on the back of the six-well plate lid. Wash twice with PBS and add fresh culture medium for photographing. Take photos at the same time every day thereafter to observe cell migration and healing ability.

[0049] 1.9 Transwell Experiment For cell counting, 600 μL of medium containing 15% FBS is typically added to the lower chamber of a 24-well plate. Then, a Transwell chamber is placed in the 24-well plate, and 200 μL of a mixture of cell suspension and complete medium is slowly added to the upper chamber. The plate is then incubated for 36 hours. Afterward, the chamber is removed, the medium is aspirated, and the cells in the upper chamber are gently wiped with a cotton swab. 1 mL of 4% paraformaldehyde is added for fixation for 30 minutes. The cells are then stained with 0.1% crystal violet for 30 minutes to remove any unbound crystal violet for subsequent microscopic examination. After air-drying, five fields of view are selected under a microscope for cell observation and counting.

[0050] 2. Animal experiments Twelve SPF-grade female Balb / c nude mice, aged 4-6 weeks and weighing 18-20g, were selected and divided into two groups of six. Both groups were injected subcutaneously into the left hind leg with 5×10 6 One SiHa empty cell. A group of tumors with a volume of 80 mm. 3 Kaempferol was administered starting at a time, once every two days. After 12 treatments, all mice were euthanized, and the tumors were removed from the nude mice for photographing. (Tumor volume calculation: width of the side) 2(×length of the longer side×0.5) 2.1 Immunohistochemical experiment on tumors in nude mice After removing the tumor from the nude mouse, it was fixed with 4% paraformaldehyde and stored in a 4°C refrigerator. Then, it was embedded in paraffin and sectioned, dewaxed and hydrated, antigen retrieval, inactivation, blocking, primary antibody incubation, secondary antibody incubation, staining and development, counterstaining, dehydration, and mounting.

[0051] 3. Results 3.1 Database data shows that CDH18 and β-catenin have a certain correlation. A comprehensive analysis of molecules interacting with CDH18 was performed using bioinformatics STRING, identifying 10 related genes and constructing a core cross-reference map. The results showed that CTNNB1, CDH6, CDH10, CDH12, CDH2, CDH9, and CDH7 had high correlations with CDH18, with CTNNB1 (β-catenin) exhibiting the highest correlation. Figure 15 A). Subsequently, CO-IP was used to verify its binding affinity, confirming the bioinformatics prediction ( Figure 15 B), and the molecular docking diagram and binding energy of CDH18 and β-catenin were analyzed using the bioinformatics ZDOCK algorithm. Figure 15 (C, 15D). Meanwhile, β-catenin is a key protein in the Wnt signaling pathway. Given that CDH18 is a transmembrane protein, it is speculated that CDH18 can participate in the Wnt pathway by binding to β-catenin on the cell membrane, thereby affecting the biological function of cervical cancer cells.

[0052] 3.2 Kaempferol inhibits in a dose-dependent manner CDH18 Gene expression We used a bioinformatics CTD website to predict kaempferol ( Figure 16 A) Can inhibit CDH18 Gene expression was analyzed, and the binding affinity between the two was determined. According to literature, a docking score less than -5.0 and greater than -7.0 indicates that the small molecule compound has a good binding ability to the target. Figure 16 B). Molecular docking of CDH18 and kaempferol was performed using the bioinformatics software ZDOCK. Figure 16 C) We added different concentrations of kaempferol to SiHa and HeLa cells, setting concentration gradients of 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM, and time gradients of 0 h, 24 h, 48 h, 72 h, and 96 h. MTT assays showed that the survival rate of both SiHa and HeLa cells decreased in a concentration-dependent manner, and the IC50 of kaempferol on SiHa and HeLa cells was determined. 50 The concentrations were 15.19 μmol / L and 35.47 μmol / L, respectively. Figure 16 D). Meanwhile, Western blot results also confirmed that kaempferol can significantly inhibit the activity of cervical cancer cells SiHa and HeLa. CDH18 Protein expression level ( Figure 16 E-16F P <0.5, P <0.01; P <0.001).

[0053] 3.3 Kaempferol-targeted inhibition CDH18 Genes thus affect the activity of the Wnt pathway. To more comprehensively verify whether kaempferol works by inhibiting... CDH18 Genes influence the Wnt / β-catenin signaling axis, thereby mitigating the development of cervical cancer. We studied the overexpression of... CDH18 Kaempferol was added to SiHa and HeLa cells at concentrations of 15.19 μmol / L and 35.47 μmol / L, respectively, to form a control group, an overexpression group, and a treatment group. The results showed that kaempferol eliminated the protein expression levels of β-catenin, TCF4, and c-Myc, while enhancing the expression level of GSK-3β and the phosphorylation level of β-catenin in a concentration-dependent manner. Figure 17 , Figure 18 (A-18E) P <0.5, P <0.01; P <0.001). Therefore, it can be concluded that kaempferol reversed [the effect]. CDH18 Gene activation of the Wnt pathway can mitigate the progression of cervical cancer.

[0054] 3.4 Kaempferol's ability to inhibit the proliferation of cervical cancer SiHa and HeLa cells in vitro Overexpressing cells were treated with kaempferol at concentrations of 15.19 μmol / L and 35.47 μmol / L, respectively. CDH18 Western blot analysis of CDK4 protein expression levels in SiHa and HeLa cells showed a significant decrease in CDK4 expression. Figure 19 (A, 19B) P <0.5, P <0.01). Simultaneously, the effect of kaempferol on cell viability was detected using the EdU assay. The results showed that kaempferol significantly reduced... CDH18 The promoting effect of genes on the proliferation of SiHa and HeLa cells ( Figure 19 (C, 19D) P<0.5. Cell proliferation rate was detected at 0h, 24h, 48h, 72h, and 96h using the MTT assay. Results showed that the cell proliferation rate in the drug-treated group was significantly lower than that in the NC control group and the overexpression group. Figure 19 E)( P <0.5, P <0.01; P The result (<0.0001) indicates that kaempferol has certain potential anti-tumor activity in cervical cancer cells, further clarifying its potential as an anti-cancer drug.

[0055] 3.5 Kaempferol induces apoptosis in cervical cancer SiHa and HeLa cells in vitro. To further determine whether kaempferol participates in regulating apoptosis in cervical cancer SiHa and HeLa cells, Western blot was used to detect the protein expression levels of apoptosis-related molecules, identifying pro-apoptotic molecule Bax and anti-apoptotic molecule Bcl-2, caspase 3, and cleaved caspase 3. Caspase 3 is the most important terminal cleavage enzyme in apoptosis, and caspase 3 is a positive apoptotic molecule. The results showed that caspase 3 expression level remained unchanged, Bax and cleaved caspase 3 expression levels increased, and Bcl-2 expression level decreased. Figure 20 A-20B) P <0.5, P <0.01; P <0.001). Furthermore, Hoechst 33258 cell nuclear staining assays were used to detect kaempferol overexpression. CDH18 The number of apoptotic cells after SiHa and HeLa gene expression was measured. We observed enhanced nuclear staining and brighter fluorescence in apoptotic cells, which exhibited round, condensed, or clumped structures. Non-apoptotic cell nuclei showed varying degrees of fluorescence intensity. Results showed that the drug-treated group had the highest number of apoptotic cells, followed by the NC control group. Overexpression of the gene... CDH18 The gene has the lowest number of apoptotic cells ( Figure 21 This leads to the conclusion that kaempferol can induce apoptosis in cervical cancer SiHa and HeLa cells, and further clarifies that kaempferol works by reducing... CDH18 Gene expression promotes apoptosis.

[0056] 3.6 Inhibitory effect of kaempferol on the migration ability of cervical cancer cells in vitro The effect of kaempferol on the migration ability of cervical cancer cells was further evaluated using wound healing assays and Transwell assays. Results showed that the kaempferol-treated group, compared to the group overexpressing [a specific type of cancer cell], [achieved better results]. CDH18The genome shows a significantly larger scratch area, weaker lateral cell healing ability, and lower migration rate. Figure 22 (A, 22B) P <0.01; P <0.001). Furthermore, the Transwell assay was used to further investigate the effect of kaempferol on cell membrane penetration ability, revealing that kaempferol also had a significant dose-dependent inhibitory effect on cell membrane penetration ability (…). Figure 22 C, 22D) P <0.5, P <0.01).

[0057] 3.7 Kaempferol can reduce tumorigenicity in nude mice in in vivo experiments. Based on the above experimental results, it can be seen that kaempferol can inhibit [the activity of other substances] in vitro. CDH18 The expression of genes inhibits the Wnt pathway, thereby slowing the development of cervical cancer. To verify whether kaempferol has the same inhibitory effect on tumor growth in vivo, we established a nude mouse tumorigenesis model. Figure 23 A) Nude mice were divided into two groups of six: an empty SiHa cell group and an empty SiHa cell-treated group. Simultaneously, a large dish of SiHa cells was injected subcutaneously into the left hind leg of each mouse. In the treated group, the tumor volume reached 80-100 mm². 3 Starting on day 26, 100 µL of 0.1 μM kaempferol was injected peritumorally every two days, and the difference in tumor volume between the group and the empty SiHa cell group was observed. Figure 23 B). Consistent with expected results, the tumor volume and growth rate in the nude mice treated with empty SiHa cells were significantly smaller than those in the empty cell group (B). Figure 23 C, 23D) P <0.5, P <0.01; P <0.001). Therefore, it can be concluded that kaempferol also has the same inhibitory effect on tumor growth in vivo.

[0058] 3.8 Kaempferol affects tumorigenesis in nude mice by inhibiting the Wnt signaling pathway. To further explore the efficacy of kaempferol in inhibiting tumor formation in nude mice via the Wnt pathway, tumor tissue was extracted from the subcutaneous tissue of nude mice and subjected to immunohistochemical staining. The protein expression levels of pathway-related molecules such as CDH18, β-catenin, GSK-3β, c-Myc, and TCF4 were evaluated. The results showed that the tumor tissue of the treated group effectively inhibited the expression of β-catenin, c-Myc, and TCF4 molecules. Figure 24 (A, 24B) P <0.5, PA concentration <0.01) can inhibit the Wnt pathway and hinder tumor formation in nude mice, demonstrating that kaempferol has good therapeutic effects on tumors in nude mice and can significantly slow down tumor growth. In vivo and in vitro experiments show that kaempferol promotes apoptosis of cervical cancer cells and inhibits the expression levels of Wnt signaling pathway-related molecules by reducing CDH18 expression levels, thereby inhibiting the proliferation and migration of cervical cancer cells.

[0059] In summary, the present invention provides... CDH18 The application of genes as targets in screening drugs for the prevention or treatment of cervical cancer, wherein the drugs are... CDH18 Targeting genes to suppress or silence them CDH18 Gene expression. Overexpression. CDH18 Genes can enhance the proliferation and migration of cervical cancer cells by regulating the Wnt pathway. CDH18 Downregulation of the gene inhibited β-catenin expression; knockdown... CDH18 Genes can inhibit the proliferation and migration of cervical cancer cells by regulating the Wnt signaling pathway. Overexpression of [the gene / gene / etc.] CDH18 Adding XAV939 to SiHa and HeLa cells significantly eliminated the gene. CDH18 The promoting effect of genes on the proliferation and migration of cervical cancer cells. MTT results showed that the survival rate of both SiHa and HeLa cells decreased in a concentration-dependent manner, and the IC50 of kaempferol on SiHa and HeLa cells was determined. 50 The concentrations were 15.19 μmol / L and 35.47 μmol / L, respectively. Meanwhile, Western blot results also confirmed that kaempferol could significantly inhibit the activity of kaempferol in cervical cancer cells SiHa and HeLa. CDH18 Protein expression levels. Kaempferol reversed [the effect]. CDH18 Gene activation of the Wnt pathway has a mitigating effect on the progression of cervical cancer. Furthermore, kaempferol exhibits potential anti-tumor activity in cervical cancer cells, further clarifying its potential as an anti-cancer drug. Kaempferol can induce apoptosis in cervical cancer SiHa and HeLa cells, further demonstrating that kaempferol works by reducing... CDH18 Gene expression promotes apoptosis. Transwell assays were used to further investigate the effect of kaempferol on cell membrane penetration, revealing a significant dose-dependent inhibitory effect. Kaempferol also exhibits similar tumor-inhibiting effects in vivo. Kaempferol demonstrates good therapeutic efficacy against tumors in nude mice, significantly slowing tumor growth. In vivo and in vitro experiments show that kaempferol promotes apoptosis in cervical cancer cells and inhibits their proliferation and migration by reducing CDH18 expression levels and thereby inhibiting the expression levels of molecules related to the Wnt signaling pathway.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. CDH18 The application of genes as targets in screening drugs for the prevention or treatment of cervical cancer, wherein the drugs are... CDH18 Targeting genes to suppress or silence them CDH18 Gene expression.

2. Inhibition CDH18 Application of gene expression reagents in the preparation of drugs for the prevention or treatment of cervical cancer.

3. Inhibition CDH18 Application of gene-expressing drugs in the preparation of drugs for the prevention or treatment of cervical cancer.

4. The application as described in claim 2 or 3, characterized in that, The inhibition CDH18 Reagents or inhibitors of gene expression CDH18 Drugs for gene expression include one or more of the following: shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, compounds, peptides, and antibodies.

5. The application as described in claim 3, characterized in that, The drug in question is kaempferol.

6. CDH18 Application of genes in inhibiting the replication of cervical cancer cells.

7. An anti-cervical cancer drug composition, characterized in that, The composition includes CDH18 Inhibitors of genes or proteins, or CDH18 A combination of gene or protein inhibitors and antitumor drugs.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. A kind CDH18 Gene knockout cell lines are characterized by, The cell line described is created using gene editing technology. CDH18 Obtained by gene knockout.

10. Application of kaempferol in the preparation of drugs for the prevention or treatment of cervical cancer.

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