Drugs that inhibit colorectal cancer metastasis by targeting glucocorticoid receptor GR

Abstract

The present invention belongs to the field of biomedicine technology and discloses a drug that inhibits colorectal cancer metastasis by targeting glucocorticoid receptor GR. The drug that inhibits colorectal cancer metastasis by targeting glucocorticoid receptor GR is 0.1 μM glucocorticoid and / or 0.1 μM belinostat. The present invention verifies the actual effect of small molecule drugs in CRC by in vitro cell invasion experiments and transcriptome sequencing analysis methods, and the effect of combined treatment with glucocorticoid and belinostat on the invasion of CRC cell line HCT116 cells; HCT116 cells are treated with 0.1 μM glucocorticoid and / or 0.1 μM belinostat for 48 hours, stained with 0.1% crystal violet and photographed, eluted with 33% acetic acid, and the absorbance value is detected by a microplate reader at 590 nm. The experimental results of the present invention show that GC promotes CRC cell invasion, and belinostat combined treatment can effectively inhibit the invasive effect caused by GC.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a drug for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR. Background Art

[0002] Currently, colorectal cancer (CRC) is the second leading cause of cancer death worldwide. In China, the incidence of colorectal cancer has been increasing. Currently, surgery, radiotherapy and chemotherapy are still the main treatments for CRC, but the significant side effects of the drugs can cause damage to organs and physiological systems. In recent years, with the rapid development of multi-omics technology, targeted therapy specifically acts on key targets in tumor occurrence and development and their downstream signaling pathways. Usually, corresponding targeted drugs are used to inhibit CRC by targeting single mutation sites such as KRAS, BRAF, EGFR, MEK, etc., which are common in cancers such as CRC. However, the current number of targeted drugs is still small, and targeted drug strategies are limited. Therefore, it is very necessary to further develop new strategies to improve the effect of targeted therapy, improve patient survival rate and quality of life after treatment.

[0003] Currently, the development of new drugs is extremely difficult, including long cycles, high costs, and high failure rates at each stage. Therefore, reusing existing drugs with corresponding indications to treat other types of diseases is becoming an increasingly promising drug development method, commonly known as "drug repurposing". Compared with the development of new drugs, "drug repurposing" has the following advantages: (1) a higher success rate, as marketed drugs have undergone systematic safety evaluation; (2) a shorter development cycle, as marketed drugs have comprehensive preclinical pharmacokinetic and toxicological parameters, and the development of new indications based on previous research can greatly shorten the development cycle; (3) a lower development cost, as compared with the development of new drugs for the same indication, "new uses of old drugs" can significantly save development costs in preclinical and clinical phases I and II.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows:

[0005] (1) Currently, surgery, radiotherapy, and chemotherapy are still the main treatments for colorectal cancer. However, due to poor drug selectivity, they have significant side effects that can cause damage to organs and physiological systems.

[0006] (2) There are still few targeted drugs available for inhibiting colorectal cancer, and the combination strategies of targeted drugs are limited. The current probability of resistance to targeted drugs is 100%. Once resistance occurs, the disease will deteriorate rapidly.

[0007] (3) Currently, the research and development of new drugs is extremely difficult, including long cycles and high costs at each stage and a high failure rate. Summary of the Invention

[0008] In response to the problems existing in the prior art, the present invention provides a drug for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR.

[0009] The present invention is achieved by a method for screening drugs that inhibit colorectal cancer metastasis by targeting the glucocorticoid receptor (GR). This method includes: reclassifying colorectal cancer patients by analyzing publicly available tumor phenotypic data, screening for new targets, and verifying their efficacy; obtaining a set of shared target genes downstream of the new targets through chromatin immunoprecipitation high-throughput sequencing data analysis; and then utilizing the CMap database to obtain and screen small molecule drugs corresponding to the target gene set.

[0010] Furthermore, the drug screening method for inhibiting colorectal cancer metastasis by targeting the glucocorticoid receptor GR also includes: screening the pathways and target proteins GR in CRC metastasis, analyzing and verifying the GR protein expression level and the effect of the protein complex formed by GR and DNA hydroxymethylation modification enzyme TET on CRC metastasis; analyzing the GR&TET&CRC target gene spectrum through ChIP-seq; and then matching the GR&TET target genes with small molecule drugs through the CMap database to determine the final small molecule drugs.

[0011] Furthermore, the method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR comprises the following steps:

[0012] Step 1: Explore targets related to CRC cancer cell metastasis and verify their effectiveness; classify CRC patients using the new target GR and analyze the correlation between GR and patient survival rates using the TCGA database;

[0013] Step 2: Using chromatin immunoprecipitation high-throughput sequencing data analysis, we obtained the downstream common target gene set of GR and TET ChIP-seq target genes in CRC cell lines (HCT116 & HT29) that overlapped with CRC differentially expressed gene DEGs;

[0014] Step 3: Use the CMap database to match the small molecule drug set corresponding to the target gene set;

[0015] Step 4: Use interaction matching to obtain target genes corresponding to small molecule drugs that have a direct effect on the target GR&TET, and screen out the small molecule drugs with the most outstanding effects;

[0016] Step 5: Verify the actual effect of small molecule drugs in CRC through in vitro cell invasion experiments and transcriptome sequencing analysis.

[0017] Furthermore, in step 1, we explored targets related to cancer cell metastasis, verified their effectiveness, classified CRC patients using new targets, and analyzed their correlation with patient survival rates using the TCGA database.

[0018] (1) Background and literature review: Glucocorticoid receptor (GR) is associated with CRC metastasis, and hypoxic environment promotes cancer cell metastasis. DNA hydroxymethylase TET family proteins promote CRC cell metastasis.

[0019] (2) GR and TET can form a protein complex to play a regulatory role in CRC metastasis, and experiments have shown that different concentrations of glucocorticoid GC promote the migration of HCT116 cells with high GR expression, but have no significant effect on HT29 cells with low GR expression; the experimental concentrations are 0.1μM and 1μM, and the action time is 48h and 72h;

[0020] (3) CRC patients in the TCGA public database were classified using GR. Analysis showed that high GR expression was associated with low survival rates in CRC patients. Combined with survey and experimental results, drugs targeting the GR-TET protein complex were screened.

[0021] Furthermore, in step 2, using chromatin immunoprecipitation sequencing high-throughput data analysis, the downstream common target gene set that overlaps with CRC differentially expressed genes (DEGs) was obtained, including:

[0022] (1) Obtain the public CHIP-seq data of GR, TET2, and TET3 of the model cell HEK293T, and intersect the target genes of GR with the target genes of TET2 or TET3 to obtain a new GR-TETs common target gene set;

[0023] (2) The three target gene sets were intersected with the differentially expressed genes DEGs of CRC to obtain the common target gene sets CRC&GR&TET2 and CRC&GR&TET3 related to CRC. Among them, the number of common target genes in CRC&GR&TET2 was larger and the interaction relationship was stronger.

[0024] Furthermore, in step 3, the small molecule drugs corresponding to the target gene set matched by the CMap database include:

[0025] The CRC&GR&TET2 target gene set obtained in step 2 was analyzed using the CMap database to obtain a small molecule drug set, which was further screened to obtain epigenetic small molecule drugs.

[0026] (1) The small molecule drug collection obtained by screening the CMap database was scored, and drugs with a norm_cs score greater than 1.5 were considered to have significant effects.

[0027] (2) Screening is performed based on the action pathways and target genes of the small molecule drug set in step (1), and the epigenetic small molecule drugs obtained are mainly HDAC broad-spectrum inhibitors.

[0028] Furthermore, in step four, the target genes of all epigenetic small molecule drugs as well as GR and TET2 were input into the STRING database platform for interaction matching. The target genes corresponding to small molecule drugs that have a direct interaction with both GR and TET2 were obtained through analysis. Among them, histone deacetylases HDAC1, HDAC2 and HDAC6 are the three most important directly associated genes. A total of three HDAC broad-spectrum inhibitors, including belinostat, target these three main associated genes.

[0029] Another object of the present invention is to provide a method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR, wherein the drug obtained by screening is 0.1 μM glucocorticoid and / or 0.1 μM belinostat.

[0030] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0031] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving these problems, we closely combine the technical solutions to be protected by the present invention and the results and data during the research and development process, and conduct a detailed and in-depth analysis of how the technical solutions of the present invention solve the technical problems and some creative technical effects brought about by solving the problems. The specific description is as follows:

[0032] The present invention verifies the effect by mining targets related to cancer cell metastasis, classifies CRC patients through the new target GR and analyzes its correlation with patient survival rate through the TCGA database; then obtains a downstream common target gene set that overlaps with CRC differentially expressed genes (DEGs) through chromatin immunoprecipitation sequencing (ChIP-seq) high-throughput data analysis; and then uses the CMap database to match small molecule drugs corresponding to the target gene set. The present invention matches the small molecule drug target gene set with GR, TET2 and TET3 in the STRING database for interaction, obtains the corresponding target genes of small molecule drugs that have a direct effect on the GR-TET protein complex, and further screens the small molecule drugs with the most outstanding effect; finally, the actual effect of small molecule drugs in CRC is verified by in vitro cell invasion experiments and transcriptome sequencing analysis.

[0033] The present invention verifies the actual effect of small molecule drugs in CRC by using in vitro cell invasion experiments and transcriptome sequencing analysis methods; the effect of combined treatment with glucocorticoids and belinostat on the invasion of CRC cell line HCT116 cells; HCT116 cells were treated with 0.1μM glucocorticoids and / or 0.1μM belinostat for 48 hours, stained with 0.1% crystal violet, photographed, eluted with 33% acetic acid, and the absorbance value was measured at 590nm by a microplate reader; the results showed that GC promoted CRC cell invasion, while belinostat combined treatment effectively inhibited the invasion caused by GC. The present invention uses transcriptome sequencing to analyze the effect of belinostat on targeting GR in CRC. The upper and lower layers of the treated cells were collected separately, total cell RNA was extracted, micro-libraries were constructed using the SMART-SEQ method, and high-throughput transcriptome sequencing was performed using the PE150 strategy to analyze the results.

[0034] The present invention reclassifies CRC patients by analyzing publicly available phenotypic data, searches for new targets, and verifies their effects; obtains a downstream shared target gene set through high-throughput data analysis using chromatin immunoprecipitation sequencing (ChIP-seq); and then utilizes the CMap database to obtain and screen small molecule drugs corresponding to the target gene set; among them, CRC patients with high GR expression (reclassified, with GR as a potential target) have a low survival rate (high metastasis rate) (experiments have shown that hypoxia can promote CRC metastasis (hydroxymethylase TET protein promotes HT29 metastasis)), and targets GR-TET protein complex drugs for drug screening to achieve drug reuse (new uses of old drugs).

[0035] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are described in detail as follows:

[0036] This study reclassifies CRC patients to identify the primary pathway and protein involved in CRC metastasis, GR, and analyzes the specific role of GR in forming a protein complex with TET. ChIP-seq is then used to analyze the GR, TET, and CRC target gene profiles. Small-molecule drug matching is performed against GR and TET target genes using the CMap database. Furthermore, the STRING database is used to identify small-molecule drug target genes that directly interact with GR and TET2, ultimately resulting in the most effective small-molecule drug. This study provides new insights into broadening the range of drug options for CRC and significantly shortens the time from drug discovery to clinical translation, demonstrating its significant clinical therapeutic potential.

[0037] Third, as auxiliary evidence for the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0038] The expected benefits and commercial value of the technical solution after transformation are as follows: This transformation can be used to treat CRC patients with high GR expression, reducing drug dosage on top of conventional targeted drug therapy and supplementing clinical treatment with drugs screened by this invention. This can further improve the survival and quality of life of patients with GR-high CRC, and is expected to yield significant benefits in clinical medication use. This novel approach and concept for developing new uses for established drugs has significant commercial value and can be applied to other similar drug development processes, driving industry development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0040] Figure 1 This is a flow chart of a method for screening drugs that inhibit colorectal cancer metastasis by targeting glucocorticoid receptor GR, provided in an embodiment of the present invention;

[0041] Figure 2 This is a schematic diagram showing that dexamethasone promotes the migration of HCT116 cells with high GR expression and HT29 cells with low GR expression, as provided in an embodiment of the present invention;

[0042] Figure 3 Schematic diagram of the effect of belinostat on the invasion of HCT116 cells induced by dexamethasone provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0044] In response to the problems existing in the prior art, the present invention provides a drug for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR. The present invention is described in detail below with reference to the accompanying drawings.

[0045] In order to enable those skilled in the art to fully understand how to implement the present invention, this section provides an explanatory embodiment that expands on the technical solutions of the claims.

[0046] like Figure 1 As shown, the method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR provided by the embodiment of the present invention comprises the following steps:

[0047] S101: Reclassify CRC patients by analyzing publicly available phenotypic data and identify novel targets significantly associated with CRC patient survival and CRC cancer cell metastasis;

[0048] S102, obtain downstream common target gene set by chromatin immunoprecipitation sequencing high-throughput data analysis;

[0049] S103, using the CMap database, obtaining and screening small molecule drugs corresponding to the target gene set.

[0050] As a preferred embodiment, the method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR provided in the embodiment of the present invention specifically comprises the following steps:

[0051] To explore targets related to cancer cell metastasis and verify their effects, we used the new target GR to classify CRC patients in the TCGA database and analyze its correlation with patient survival. Then, through high-throughput data analysis of chromatin immunoprecipitation sequencing (ChIP-seq), we obtained a set of downstream common target genes that overlapped with CRC differentially expressed genes (DEGs). We then used the CMap database to match small molecule drugs corresponding to the target gene set. We matched the small molecule drug target gene set with GR&TET2 in the STRING database for interaction, obtained target genes corresponding to small molecule drugs that had a direct effect on GR&TET2, and further screened the small molecule drugs with the most outstanding effects. Finally, we verified the actual effect of small molecule drugs in CRC through in vitro cell invasion experiments and transcriptome sequencing analysis.

[0052] The present invention screens the main pathways and proteins involved in CRC metastasis, analyzes the specific role of GR protein, and forms a protein complex with TET. ChIP-seq is used to analyze the GR & TET & CRC target gene spectrum, and small molecule drug matching is performed on GR & TET target genes using the CMap database. The specific process is as follows:

[0053] 1. Explore the relationship between GR and cancer cell metastasis, verify the effect, classify CRC patients in the TCGA database using the new target GR and analyze its correlation with patient survival rate;

[0054] 1.1 Background & Literature Review: Glucocorticoid receptor (GR) is associated with CRC metastasis, and hypoxia promotes cancer cell metastasis. Hydroxymethylated TET promotes CRC cell metastasis.

[0055] 1.2GR and TET can form a protein complex to exert regulatory effects, and experiments have shown that different concentrations of glucocorticoid (GC) promote the migration of HCT116 cells with high GR expression, but have no significant effect on HT29 cells with low GR expression (concentration: 0.1μM & 1μM; duration: 48 & 72h);

[0056] 1.3 CRC patients in the TCGA public database were classified using GR. Analysis showed that high GR expression was associated with low survival rates in CRC patients. Therefore, based on the above survey and experimental results, drugs targeting the GR-TET protein complex were screened to achieve drug reuse, which may have an inhibitory effect on CRC metastasis to a certain extent.

[0057] 2. Chromatin immunoprecipitation sequencing (ChIP-seq) high-throughput data analysis to obtain a set of downstream common target genes that overlap with CRC differentially expressed genes (DEGs);

[0058] 2.1 Obtain the public CHIP-seq data of GR, TET1, TET2, and TET3 of the model cell HEK293T, and intersect the target genes of GR with the target genes of TET1, TET2, or TET3 to obtain a new GR-TETs common target gene set;

[0059] 2.2 The above three target gene sets were intersected again with the differentially expressed genes (DEGs) of CRC to obtain the common target gene sets related to CRC (CRC&GR&TET1, CRC&GR&TET2, CRC&GR&TET3). Analysis found that the number of common target genes of CRC&GR&TET2 was larger and the interaction relationship was stronger.

[0060] 3. Use the CMap database to match small molecule drugs corresponding to the target gene set;

[0061] 3.1 Analyze the CRC, GR, and TET2 target gene sets obtained in 2.2 using the CMap database to obtain a small molecule drug set (norm_cs score greater than 1.5);

[0062] 3.2 Further screening of epigenetic small molecule drugs, mainly HDAC broad-spectrum inhibitors, etc.

[0063] 4. All target genes of epigenetic small molecule drugs, as well as GR and TET2, were entered into the STRING database platform for interaction matching. Analysis revealed target genes for small molecule drugs with direct interactions with both GR and TET2. Histone deacetylases HDAC1, HDAC2, and HDAC6 were the most prominent directly associated genes. Three broad-spectrum HDAC inhibitors, including Belinostat, target these three key genes.

[0064] The present invention verifies the actual effect of small molecule drugs in CRC by using in vitro cell invasion experiments and transcriptome sequencing analysis methods; the effect of combined treatment with glucocorticoids and belinostat on the invasion of CRC cell line HCT116 cells; HCT116 cells were treated with 0.1μM glucocorticoids and / or 0.1μM belinostat for 48 hours, stained with 0.1% crystal violet, photographed, eluted with 33% acetic acid, and the absorbance value was detected by a microplate reader at 590nm; the results showed that GC promoted CRC cell invasion, while co-treatment with Belinostat effectively inhibited the invasion caused by GC. The present invention uses transcriptome sequencing to analyze the effect of belinostat on targeting GR in CRC. The upper and lower layers of the treated cells were collected separately, total cell RNA was extracted, micro-libraries were constructed using the SMART-SEQ method, and high-throughput transcriptome sequencing was performed using the PE150 strategy, and the results were analyzed.

[0065] In order to prove the creativity and technical value of the technical solution of the present invention, this section provides application examples of the claimed technical solution on specific products or related technologies.

[0066] Example 1: Mining targets related to CRC cancer cell metastasis; verifying their inhibitory effects on cell migration, classifying CRC patients by new targets, and analyzing their correlation with patient survival rates through the TCGA database.

[0067] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0068] (1) Analysis of The Cancer Genome Atlas (TCGA) database: Survival analysis of GR expression and overall survival (OS) in patients with colon adenocarcinoma (n=512) showed that patients with low levels of GR in target tissues had a higher OS rate compared with patients with high GR.

[0069] (2) Cell line procurement: Human colorectal cell line HT-29 was purchased from the American Type Culture Collection (ATCC), and HCT-116 was purchased from the China Type Culture Collection (CCTCC).

[0070] (3) Prepare complete culture medium: DMEM / F12 medium, 10% fetal bovine serum, 1% GlutaMAX, and 0.1% mycoplasma preventive agent. Filter the complete culture medium through a 0.22 μm sterile filter and store at 4°C until ready for use. Re-prepare after no more than one month.

[0071] (4) Cell migration assay: 5×10 4HCT116 or HT29 cells were resuspended in 200 μL of DMEM / F-12 and treated with different doses of dexamethasone (0, 0.1, or 1 μM) and seeded into the upper chamber of an 8.0 mm Transwell cell culture insert. 800 μL of DMEM / F-12 supplemented with 10% FBS was added to the bottom chamber as a chemoattractant. The cells were then incubated in a humidified incubator at 37°C with 5% CO2 for 48 or 72 hours.

[0072] The cells on the upper inner layer of the chamber were gently removed with a moistened cotton swab, and then washed three times with DPBS for 5 minutes each. The cells on the lower surface of the membrane were stained with 0.1% crystal violet for 10 minutes, and then the chamber was washed twice with DPBS and air-dried.

[0073] Under bright-field microscopy, observe and photograph cells migrating to the lower layer. Cell-bound crystal violet was eluted with 400 μL of 33% acetic acid and shaken on a horizontal mixer for 10 minutes. The crystal violet eluate from the lower layer was transferred to a 96-well microplate, and the absorbance at 590 nm was measured using a microplate reader. All experiments were repeated three times.

[0074] Example 2 Chromatin immunoprecipitation sequencing (ChIP-seq) high-throughput data analysis was performed to obtain a set of downstream common target genes that overlapped with CRC differentially expressed genes (DEGs).

[0075] (1) Sample pretreatment for ChIP-seq: About 3×10 7 Cells (HCT116 and HT29 cells) were cross-linked with 1% formaldehyde in PBS for 15 minutes in a 37°C incubator and then lysed with lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, 1× PIC). Chromatin DNA was precipitated with 4 μg of specific antibody (TET1, TET2, or TET3), eluted, and cross-links reversed. DNA was purified using the Qiaquick PCR purification kit according to the manufacturer's recommendations. All chromatin DNA samples were run in duplicate.

[0076] (2) ChIP-seq high-throughput data analysis: The raw sequencing data were first quality controlled using FastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) using default parameters. The raw data were then aligned to the unmasked human reference genome (GRCh38, hg38) using the Burrows-Wheeler Aligner (BWA) tool for paired-end alignment.

[0077] Flagstat in Samtools was used to check read mapping quality. Duplicate reads were then removed using Samtools, and only sequences with a mapping quality score > 15 were retained. Remaining sequences with specific binding peaks for GR and TET1 / 2 / 3 were identified using MACS v2 (Model-Based ChIP Sequencing Analysis) using default settings for all paired-end data.

[0078] The genomic distribution of GR and TET1 / 2 / 3 binding sites was analyzed using GREAT Version 4 (Genomic Region Enrichment Annotation Tool) with the aid of the hg38 genome annotation, and the 1000 bp upstream and downstream of the transcription start site (TSS) were considered as promoter regions.

[0079] Comprehensive motif screening was performed using the MEME suite within 100 bp of the center of the GR and TET2 binding peaks. Motifs within gene peaks were searched using the Large Nucleotide Dataset Motif Analysis (MEME-ChIP) scanner. Biological process ontology analysis of annotated genes was performed using Metascape.

[0080] Example 3 uses the CMap database to match small molecule drugs corresponding to the target gene set.

[0081] (1) The 46 GR candidate genes and 89 TET2 candidate genes in HCT116 cells and the 240 GR and TET2 common genes in HEK293T cells were divided into two parts based on DEGs, namely up-regulated genes and down-regulated genes (Ab Foldchange>=1.5), and applied to CMap analysis.

[0082] (2) Based on the normalized connectivity score (norm_cs>=1.5 in at least one list and >=1.0 in the other lists), two candidate target gene lists, one HCT116 cell list (co-target genes of GR and TET2), and one HEK293T cell list (co-target genes of GR and TET2) were selected, and a total of 97 co-target compounds with the same effect were screened.

[0083] The embodiments of the present invention have achieved some positive results during the development or use process, and indeed have great advantages over the existing technology. The following content describes them in conjunction with data, charts, etc. from the experimental process.

[0084] like Figure 2 As shown in the cell migration assay, dexamethasone promoted the migration of HCT116 cells with high GR expression, but had no significant effect on HT29 cells with low GR expression. Furthermore, survival analysis showed that the survival rate of CRC patients with high GR expression was significantly reduced.

[0085] like Figure 3As shown in the figure, cell invasion assay showed that belinostat had a significant inhibitory effect on the invasion of HCT116 cells induced by dexamethasone.

[0086] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for screening drugs that inhibit colorectal cancer metastasis by targeting glucocorticoid receptor GR, characterized in that: include: Step 1: Explore targets related to CRC cancer cell metastasis and verify their effectiveness; classify CRC patients using the new target GR and analyze the correlation between GR and patient survival rates using the TCGA database; Step 2: Using chromatin immunoprecipitation high-throughput sequencing high-throughput data analysis, we obtained a set of downstream common target genes that overlapped with CRC differentially expressed genes (DEGs); Step 3: Use the CMap database to match the small molecule drug set corresponding to the target gene set; Step 4: Use interaction matching to obtain target genes corresponding to small molecule drugs that have direct interactions with GR&TET, and screen out the most effective epigenetic small molecule drugs; Step 5: Verify the actual effect of small molecule drugs in CRC through in vitro cell invasion assays and transcriptome sequencing analysis; In step 2, using chromatin immunoprecipitation sequencing high-throughput data analysis, we obtained a set of downstream common target genes that overlap with CRC differentially expressed genes (DEGs), including: (1) Obtain the public ChIP-seq data of GR, TET1, TET2, and TET3 of the model cell HEK293T, and intersect the target genes of GR with the target genes of TET1, TET2, and TET3 to obtain a new GR-TETs common target gene set; (2) The three target gene sets were intersected with the differentially expressed genes DEGs of CRC to obtain the common target gene sets CRC&GR&TET1, CRC&GR&TET2, and CRC&GR&TET3 related to CRC. Among them, CRC&GR&TET2 had a larger number of common target genes and a stronger interaction relationship; In step 3, the small molecule drugs that match the target gene set using the CMap database include: The CRC&GR&TET2 target gene set obtained in step 2 was analyzed using the CMap database to obtain a small molecule drug set, which was then screened to obtain epigenetic small molecule drugs.

2. The method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR according to claim 1, wherein: The drug screening method for inhibiting colorectal cancer metastasis by targeting the glucocorticoid receptor GR also includes: screening the pathways and target proteins GR in CRC metastasis, analyzing and verifying the GR protein expression level and the effect of the protein complex formed by GR and TET on CRC metastasis; analyzing the GR&TET&CRC target gene spectrum through CHIP-seq; and then matching the GR&TET target genes with small molecule drugs through the CMap database to determine the final small molecule drug.

3. The method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR according to claim 1, wherein: Step 1 involves exploring targets related to cancer cell metastasis, validating the effects, classifying CRC patients using the new target GR, and analyzing the correlation between GR and patient survival rates using the TCGA database. (1) Background and literature review: Glucocorticoid receptor (GR) is associated with CRC metastasis, and hypoxic environment promotes cancer cell metastasis, while hydroxymethylated TET promotes CRC cell metastasis; (2) GR and TET can form a protein complex to play a regulatory role in CRC metastasis, and experiments have shown that different concentrations of glucocorticoid GC promote the migration of HCT116 cells with high GR expression, but have no significant effect on HT29 cells with low GR expression; (3) CRC patients in the TCGA public database were classified using GR. Analysis showed that high GR expression was associated with low survival rates in CRC patients. Combined with survey and experimental results, drugs targeting the GR-TET protein complex were screened.

4. The method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR according to claim 1, wherein: The norm_cs score of the small molecule drug set in step three is greater than 1.5; the epigenetic small molecule drug in step four is a broad-spectrum HDAC inhibitor.

5. The method for screening drugs for inhibiting colorectal cancer metastasis by targeting glucocorticoid receptor GR according to claim 1, wherein: In step 4, the target genes of all epigenetic small molecule drugs as well as GR and TET2 were input into the STRING database platform, and the target genes directly related to both GR and TET2 were analyzed; among them, histone deacetylases HDAC1, HDAC2 and HDAC6 were three directly associated genes, and a total of three major associated genes were targeted by the three HDAC broad-spectrum inhibitors including Belinostat.

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