A potential anti-colorectal cancer drug, mining method and application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a potential anti-colorectal cancer drug, a mining method and an application. The potential anti-colorectal cancer drugs are perampanel, fosinopril and aminoglutethimide. The mining method of potential anti-colorectal cancer drugs includes: obtaining a signature target gene set by analyzing the transcriptome data of the natural small molecule compound anthocyanin for colorectal cancer treatment; and then using the CMap database to screen out the corresponding potential anti-colorectal cancer small molecule drugs. The present invention verifies the actual effect of anthocyanin in CRC and determines the optimal action conditions through in vitro cell viability experiments; transcriptome sequencing analyzes the functional pathway of anthocyanin's anti-colorectal cancer effect, and combines it with the anthocyanin receptor PPARChIP gene set, indicating that the Signature gene is the key target gene of the anthocyanin effect; matching with the PPARChIP gene set KEGG results reflects the effectiveness of the repositioning of candidate drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a potential anti-colorectal cancer drug, a mining method and an application thereof. Background Art

[0002] According to statistics, in 2020, colorectal cancer (CRC) is the third most prevalent cancer among all men, both in new cases and deaths. And more than 1.93 million people were diagnosed with colorectal cancer among all new cancer cases. Since 2000, the incidence of colorectal cancer has gradually increased, while the age of onset has gradually decreased. The reason for the increase in the number of people in the younger age group is unknown, but it may be related to dietary changes, lack of exercise and obesity. For the treatment of colorectal cancer, there are mainly surgical resection, radiotherapy and chemotherapy, and targeted drugs or immunotherapy. Despite great improvements in diagnosis and treatment methods, the prognosis of colorectal cancer remains poor. Studies have reported that managing inflammation by consuming natural small molecule compounds (foods / extracts) rich in antioxidants may be a potential strategy. Cultivating good eating habits is beneficial to intestinal health and is beneficial to fighting the inflammatory state to reduce the level of inflammation, thereby preventing the onset of CRC. Anthocyanins are considered to be promising potential therapeutic compounds because of their known antioxidant and anti-inflammatory properties, which can promote the relevant health benefits of CRC. However, due to its unstable and complex structure, low bioavailability, difficulty in extraction and high price, it is very necessary to conduct research on redesigning drugs that inhibit CRC cell growth and invasion based on anthocyanin screening.

[0003] At present, the development of new drugs is extremely difficult, including long development 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, known as "drug reuse". Compared with the development of new drugs, "drug reuse" has the following advantages: (1) a higher success rate, as marketed drugs have undergone systematic safety evaluation; (2) a short 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, "old drugs for new uses" can greatly save development costs in preclinical and clinical phases I and II.

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

[0005] Existing anthocyanins have problems such as unstable and complex structure, low bioavailability, difficulty in extraction and high price. Although they have good anti-colorectal cancer effects in cell experiments, the amount entering the blood in animal experiments is small and the utilization rate is low;

[0006] At the same time, the research and development of new drugs is extremely difficult, including long development cycles, high costs, and high failure rates. It takes an average of 8 to 10 years for new drug development from compound molecules to actual clinical use, and requires a lot of human and material support, with huge time and economic costs. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a potential anti-colorectal cancer drug, a mining method and an application, and particularly relates to a natural small molecule compound - anthocyanin, a CMap database and drug recycling.

[0008] The present invention is achieved by analyzing the transcriptome data of anthocyanin treatment of colorectal cancer to find and obtain the signature target gene set; then using the CMap database to screen out the corresponding potential small molecule drugs, which provides a new idea for broadening the range of drug selection for CRC, greatly shortens the time from drug discovery to clinical transformation, and has important clinical therapeutic significance.

[0009] The present invention provides an anti-colorectal cancer drug, and the anti-colorectal cancer drug is shown in the following table:

[0010]

[0011] Methods for discovering potential anti-colorectal cancer drugs include:

[0012] By analyzing the transcriptome data of anthocyanins in colorectal cancer, we obtained the signature target gene set; then, we used the CMap database to screen out the corresponding potential small molecule drugs against colorectal cancer.

[0013] Further, the methods for discovering potential anti-colorectal cancer drugs also include:

[0014] Through research, we found that anthocyanins, a natural small molecule compound with anti-colorectal cancer effects, were found. After treating the HT29 colorectal cancer cell line with anthocyanins, CCK8 was used to detect cell viability to obtain the effects and optimal conditions of anthocyanins. High-throughput data analysis was performed through transcriptome sequencing, and the influencing factors were evaluated. Signature gene sets were screened out through temporal analysis and differential gene analysis, and the CMap database was used to match and screen the corresponding potential small molecule drugs against colorectal cancer. Finally, cell viability experiments and transcriptome sequencing were used to verify the actual effects of small molecule drugs in colorectal cancer.

[0015] Furthermore, the method for mining potential anti-colorectal cancer drugs includes the following steps:

[0016] Step 1: Find natural small molecule compounds that have anti-colorectal cancer effects and are related to nuclear receptors, verify the anti-cancer effects, and obtain the optimal action conditions;

[0017] Step 2: Through transcriptome sequencing analysis, systematic evaluation was conducted on the effects of cyanine structure, concentration, and time on gene expression in HT29 cells;

[0018] Step 3: Screening of anthocyanin characteristic gene sets and mining of potential functional pathways;

[0019] Step 4: Obtain the signature gene set for CMap analysis through the results of time series analysis and differential gene analysis, and screen for potential colorectal cancer drugs similar to anthocyanins.

[0020] Furthermore, in step 1, the search for natural small molecule compounds with anti-colorectal cancer effects and related to nuclear receptors is conducted to verify the anti-cancer effects and obtain the optimal action conditions, including:

[0021] (1) Background and literature research;

[0022] Anthocyanidins are natural flavonoid compounds and have anti-colorectal cancer effects. PPAR is a direct receptor for anthocyanidins and has an antioxidant lipid metabolism effect.

[0023] (2) Anthocyanin treatment inhibits proliferation of colorectal cancer cell lines;

[0024] Compared with the control group, the growth inhibitory effect of the treatment group first increased and then decreased with time, and increased with the increase of concentration; and the experiment showed that under the conditions of concentration of 0.1μM, 1μM, 10μM&100μM and time of 12h, 24h, 48h&72h, the condition with the greatest growth inhibitory effect was 100μM for 12h.

[0025] Furthermore, the direct nuclear receptor of anthocyanins is PPAR and the colorectal cancer cell line is HT29.

[0026] Furthermore, in step 2, the effects of anthocyanin structure, concentration and time on gene expression in HT29 cells were systematically evaluated through transcriptome sequencing analysis, including:

[0027] (1) Transcriptome sequencing was performed on HT29 cells in the concentration, time and structure treatment groups and the control group, and the three replicate data were merged after upstream quality control;

[0028] (2) Calculate TPM values ​​to cluster and analyze the full expression profiles of genes under different structures, concentrations, and time;

[0029] (3) rank-rank hypergeometric overlap analysis of the same / opposite expression patterns among different structures, concentrations, and times;

[0030] (4) The data of the three anthocyanins at different concentrations were mean integrated and subjected to GSEA analysis to obtain the enriched functional pathways.

[0031] Furthermore, the concentrations of the treatment groups in step 2 were 1 μM and 100 μM, the treatment time was 12 h and 24 h, and the structures were no sugar group, monosaccharide group and disaccharide group.

[0032] Furthermore, the screening of anthocyanin characteristic gene sets and potential functional pathway mining in step 3 include:

[0033] (1) Time series analysis of the three anthocyanins was performed, with 8 patterns analyzed in each group, and the pattern with significant changes within 12 h was selected;

[0034] (2) The gene set with significant changes in anthocyanin 12 h was selected for KEGG pathway enrichment analysis to explore potential functional pathways;

[0035] (3) By analyzing the PPAR ChIP dataset in existing literature, hypothesis testing and KEGG analysis were performed with overlapping gene sets to determine the potential pathways of anthocyanin effects.

[0036] Furthermore, in step 4, the signature gene set for CMap analysis was obtained through the results of temporal analysis and differential gene analysis, and the potential colorectal cancer drug screening similar to anthocyanins was performed, including:

[0037] (1) The data of anthocyanin 12 h treatment were selected for differential gene analysis, and the overlap with the temporal analysis gene set was taken as the signature gene set for CMap matching analysis;

[0038] (2) Perform CMap analysis on the online website CLUE; click Tools-Query, select Geneexpression, Touchstone, Latest in Query parameters; input signature gene sets according to up and down regulation, and click SUBMIT to start matching analysis;

[0039] (3) Download the Compound result table and obtain a small molecule drug set by screening CRC cell lines with a norm_cs score ≥ 1.5; screen potential anti-colorectal cancer drugs by matching drug target_name with the PPAR ChIP gene set and investigating the anticancer type and effect of the drug in the literature;

[0040] (4) Download the shRNA and Overexpression result tables, and obtain the corresponding gene sets according to the conditions in step (3); perform KEGG analysis on the two gene sets and match them with the PPAR ChIP gene set KEGG results to verify that the signature genes highly represent the molecular mechanism of anthocyanin's cancer inhibition function.

[0041] 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:

[0042] First, the present invention verifies the actual effect of anthocyanins in CRC through in vitro cell viability experiments and explores its optimal action conditions. The transcriptome sequencing of the present invention analyzes the functional pathway of anthocyanins' anti-colorectal cancer effect, and combines it with the anthocyanin receptor PPAR ChIP gene set, indicating that the signature gene is largely the key target gene of the anthocyanin effect. The present invention uses CMap to match the Compound, shRNA and Overexpression gene sets, and reflects the effectiveness of candidate drug repositioning from literature research and matching with the PPAR ChIP gene set KEGG results.

[0043] The present invention obtains anthocyanin, a natural small molecule compound with anti-colorectal cancer, through research. After anthocyanin treatment of HT29 colorectal cancer cell line, CCK8 detects cell viability to obtain the effect and optimal action conditions of anthocyanin; then transcriptome sequencing (RNA-seq) is used for high-throughput data analysis and evaluation of influencing factors. The present invention screens out signature gene sets through temporal analysis and differential gene analysis, and then uses the CMap database to match and screen corresponding small molecule drugs; CMap is used to match knockout and overexpressed gene sets, and the KEGG enrichment pathways are basically overlapped or similar to the KEGG enrichment pathways of genes related to anthocyanin target genes, indicating that these gene features highly represent the molecular mechanism of anthocyanin inhibiting cancer, thereby reflecting the efficacy support for the repositioning of the candidate drugs obtained by screening.

[0044] Second, the method for mining potential anti-colorectal cancer drugs provided by the present invention analyzes the transcriptome data of the natural small molecule compound anthocyanin in the treatment of colorectal cancer to obtain the signature target gene set; then uses the CMap database to screen out the corresponding potential small molecule drugs, which provides a new idea for broadening the range of drug selection for CRC, greatly shortens the time from drug discovery to clinical transformation, and has important clinical treatment significance.

[0045] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0046] The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0047] Screening potential anti-colorectal cancer drugs to broaden the drug selection for the treatment of colorectal cancer. Using known anti-colorectal cancer natural small molecule anthocyanins to screen similar small molecule drugs reflects the concept of "medicine and food are of the same origin", and the screened drugs are also safer.

[0048] The present invention can also be used for other cancer drug screening applications, and according to the types of natural small molecules used and the types of matching compounds, preventive health-care small molecules with anti-cancer effects can be obtained. Health-care small molecules can be marketed without clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] 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 creative work.

[0050] Figure 1 is a flow chart of a method for mining potential anti-colorectal cancer drugs provided by an embodiment of the present invention;

[0051] Figure 2 The inhibition of three anthocyanins on colorectal cancer cells (a) is a line graph of cyanidin inhibiting the proliferation of colorectal cancer cells provided in an embodiment of the present invention, (b) is a line graph of cyanidin-3-O-glucose inhibiting the proliferation of colorectal cancer cells provided in an embodiment of the present invention, and (c) is a line graph of cyanidin-3-O-rutose inhibiting the proliferation of colorectal cancer cells provided in an embodiment of the present invention (Note: the values ​​in brackets are the P values ​​of the four treatment times and the untreated at 100 μM);

[0052] Figure 3 (a) is a KEGG rendering of a CMap-matched positively correlated Overexpression gene set provided in an embodiment of the present invention, (b) is a KEGG rendering of a CMap-matched negatively correlated Overexpression gene set provided in an embodiment of the present invention, (c) is a KEGG rendering of a CMap-matched positively correlated shRNA gene set provided in an embodiment of the present invention, and (d) is a KEGG rendering of a CMap-matched negatively correlated shRNA gene set provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution 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 used to limit the present invention.

[0054] In view of the problems existing in the prior art, the present invention provides a potential anti-colorectal cancer drug, an exploration method and an application thereof. The present invention is described in detail below in conjunction with the accompanying drawings.

[0055] In order to enable those skilled in the art to fully understand how to implement the present invention in detail, this section is an explanatory embodiment that expands and describes the technical solution of the claims.

[0056] The embodiment of the present invention obtains anthocyanin, a natural small molecule compound with anti-colorectal cancer through research. After anthocyanin treatment of HT29 colorectal cancer cell line, CCK8 detects cell viability to obtain the effect and optimal action conditions of anthocyanin. Then, high-throughput data analysis is performed through transcriptome sequencing (RNA-seq), and influencing factors are evaluated. Through temporal analysis and differential gene analysis, signature gene sets are screened out, and then the CMap database is used to match and screen corresponding small molecule drugs. Using CMap to match knockout and overexpressed gene sets, the KEGG enrichment pathways are basically overlapping or similar to the KEGG enrichment pathways of genes related to anthocyanin target genes, indicating that these gene features highly represent the molecular mechanism of anthocyanin in inhibiting cancer, thereby reflecting the efficacy support for the repositioning of the candidate drugs obtained by screening.

[0057] like Figure 1 As shown, the method for mining potential anti-colorectal cancer drugs provided by the embodiment of the present invention comprises the following steps:

[0058] S101, analysis of transcriptome data of natural small molecule compounds anthocyanins in the treatment of colorectal cancer;

[0059] S102, obtaining the signature target gene set through the analysis results of the transcriptome data;

[0060] S103, using the CMap database, screen out corresponding potential anti-colorectal cancer small molecule drugs.

[0061] As a preferred embodiment, the method for mining potential anti-colorectal cancer drugs provided in the embodiment of the present invention specifically comprises the following steps:

[0062] 1. Search for natural small molecule compounds that have anti-colorectal cancer effects and are related to nuclear receptors, verify the anti-cancer effects, and obtain the optimal action conditions.

[0063] 1.1 Background & Literature Research;

[0064] Anthocyanidins are natural flavonoid compounds and have anti-colorectal cancer effects. PPAR is the receptor of anthocyanidins and has antioxidant lipid metabolism effects.

[0065] 1.2 Anthocyanin treatment of colorectal cancer cell line (HT29) can inhibit its proliferation;

[0066] Compared with the control group, the growth inhibitory effect of the treatment group first increased and then decreased with time, and increased with the increase of concentration; and the experiment proved that the growth inhibitory effect was the largest under the conditions of 12h and 100μM (concentration: 0.1μM, 1μM, 10μM&100μM; time: 12h, 24h, 48h&72h).

[0067] 2. Transcriptome sequencing (RNA-seq) analysis was used to systematically evaluate (structure, concentration and time) the effects of anthocyanins on gene expression in HT29 cells.

[0068] 2.1 Transcriptome sequencing was performed on HT29 cells in the treatment group (concentration: 1 μM, 100 μM; time: 12 h, 24 h, structure: no sugar, monosaccharide, disaccharide) and the control group, and the three replicate data were merged after upstream quality control;

[0069] 2.2 Calculate TPM values ​​to cluster and analyze the full expression profile of genes under different structures, concentrations and time;

[0070] 2.3 Rank-rank hypergeometric overlap (RRHO) analysis showed that the expression patterns of different structures, concentrations and time were the same / opposite; there were significant differences in the transcriptional level among the three anthocyanin structures, among which the time effect had a greater impact and the concentration effect had a smaller impact;

[0071] 2.4 The data of the three anthocyanins at different concentrations were mean integrated and subjected to GSEA analysis to display the enriched functional pathways.

[0072] 3. Screening of anthocyanin characteristic gene sets and mining of their potential functional pathways.

[0073] 3.1 Time series analysis of the three anthocyanins was performed, with 8 patterns analyzed in each group, and the pattern with significant changes in 12h was selected;

[0074] 3.2 The gene sets with significant changes in anthocyanins at 12 h were selected for KEGG pathway enrichment analysis to explore potential functional pathways;

[0075] 3.3 By analyzing the PPAR ChIP datasets in existing literature, hypothesis testing and KEGG analysis were performed on overlapping gene sets with the above gene sets, indicating that these genes are largely the key target genes of anthocyanin effects.

[0076] 4. The signature gene set for CMap analysis was obtained through time series analysis and differential gene analysis results, and potential colorectal cancer drug screening similar to anthocyanins was performed.

[0077] 4.1 The data of anthocyanin 12h treatment were selected for differentially expressed genes (DEGs) analysis, and the overlap with the above-mentioned temporal analysis gene set was taken as the signature gene set for CMap matching analysis;

[0078] 4.2 Perform CMap analysis on the online website CLUE (http: / / clue.io); click Tools-Query, select Gene expression (L1000), Touchstone, Latest in Query parameters; input Signature gene set according to up and down regulation, and click SUBMIT to start matching analysis;

[0079] 4.3 Download the Compound result table, and obtain a small molecule drug set by screening CRC cell lines with norm_cs score ≥ 1.5; screen potential anti-colorectal cancer drugs by matching drug target_name with PPAR ChIP gene set and literature research on drug anticancer type and effect;

[0080] 4.4 Download the shRNA and Overexpression result tables, and obtain the corresponding gene sets through the conditions in 4.3; perform KEGG analysis on the two gene sets and match them with the PPAR ChIP gene set KEGG results to verify that the signature genes highly represent the molecular mechanism of anthocyanin's cancer inhibition function, thereby reflecting the effectiveness of candidate drug repositioning.

[0081] The present invention verifies the actual effect of anthocyanins in CRC through in vitro cell viability experiments and explores its optimal action conditions. The present invention analyzes the functional pathway of anthocyanins' anti-colorectal cancer effect through transcriptome sequencing, and combines it with the anthocyanin receptor PPAR ChIP gene set, indicating that the signature gene is largely the key target gene of the anthocyanin effect. The present invention uses CMap to match Compound, shRNA and Overexpression gene sets, and reflects the effectiveness of candidate drug repositioning from literature research and matching with the PPAR ChIP gene set KEGG results.

[0082] 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.

[0083] like Figure 1 As shown, Example 1 investigates natural small molecules against colorectal cancer and performs cell viability detection and transcriptome library construction

[0084] Literature research found that anthocyanins are natural flavonoid compounds and have anti-colorectal cancer effects. Oxisome proliferator-activated receptors are receptors for anthocyanins and have antioxidant lipid metabolism effects.

[0085] (1) Chemicals

[0086] Cyanidin chloride (CyCl), cyanidin 3-O-glucose (C3G) and cyanidin 3-O-rutose (C3R). Stock solutions of compounds were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C before use. Human colorectal cancer HT29 cell line was purchased from American Type Culture Collection (ATCC). 10% fetal bovine serum (FBS), 1% GlutaMAX, 1% penicillin / streptomycin.

[0087] (2) Cell viability determination

[0088] HT29 cells were seeded into 96-well plates at a density of 10,000 cells / well. After the cells were fully attached, they were treated with 0μM, 0.1μM, 1μM, 10μM and 100μM anthocyanins (CyCl, C3G or C3R) for 0h, 12h, 24h, 48h and 72h, respectively, in triplicate. The culture medium was replaced with fresh medium of the corresponding compound every 24 hours. The experiment was repeated 3 times.

[0089] At each time point of treatment, 10 μl of CCK8 solution was added to each well and incubated with cells for another 2 h in a 37°C incubator. Cell viability was measured at 450 nm using a SpectraMax iD3 Multi-Mode Microplate Reader. Based on the measured absorbance, the cell viability of each treatment group relative to the control group was calculated.

[0090] (3) RNA sequencing library construction and sequencing

[0091] Total RNA was extracted from each sample using the RNeasy Mini Kit according to the manufacturer's instructions. The amount of total RNA was measured using the Nanodrop 1000, and the integrity of the RNA was checked using the 2100 Bioanalyzer System.

[0092] According to the manufacturer's recommendations, use Ultra RNA Library Prep Kit for Transcriptome library construction. Briefly, 1 μg RNA from each sample was used for purification using poly-Toligo-attached magnetic beads. Fragmentation was performed using NEB Next First Strand Synthesis Reaction Buffer (5X). The first and second strands of cDNA were synthesized using transcriptase, followed by end repair by exonuclease / polymerase. After adenylation of the 3' end of the DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. Afterwards, cDNA fragments of 250-300 bp were selected by two-step purification with AMPure XP beads, PCR was performed with Phusion High-Fidelity DNA Polymerase, and 13 cycles of 98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec were performed. The products were purified with AMPure XP beads, and the library quality was assessed using the Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads.

[0093] Example 2 Transcriptome sequencing analysis and evaluation of influencing factors; screening of signature gene sets through temporal analysis and differential gene analysis

[0094] (1) Transcriptome sequencing was performed on HT29 cells in the concentration, time and structure treatment groups and the control group, and the three replicate data were merged after upstream quality control;

[0095] (2) Calculate TPM values ​​to cluster and analyze the full expression profiles of genes under different structures, concentrations, and time;

[0096] (3) rank-rank hypergeometric overlap analysis of the same / opposite expression patterns among different structures, concentrations, and times;

[0097] (4) The data of the three anthocyanins at different concentrations were mean integrated and subjected to GSEA analysis to obtain the enriched functional pathways.

[0098] (5) Time series analysis of the three anthocyanins was performed, with eight patterns analyzed in each group, and the pattern with significant changes within 12 h was selected;

[0099] (6) The gene set with significant changes in anthocyanin 12h was selected for KEGG pathway enrichment analysis to explore potential functional pathways;

[0100] (7) By analyzing the PPAR ChIP dataset in existing literature, hypothesis testing and KEGG analysis were performed with overlapping gene sets to identify the key target genes of anthocyanin effects.

[0101] (8) The data of anthocyanin 12 h treatment were selected for differential gene analysis, and the overlap with the temporal analysis gene set was taken as the signature gene set;

[0102] Example 3 Using the CMap database to match signature gene sets and screen for potential colorectal cancer drugs similar to anthocyanins

[0103] (1) Perform CMap analysis on the online website CLUE (http: / / clue.io); click Tools-Query, select Gene expression (L1000), Touchstone, Latest in Query parameters; input signature gene sets according to up and down regulation, and click SUBMIT to start matching analysis;

[0104] (2) Download the Compound result table and obtain a small molecule drug set by screening CRC cell lines with a norm_cs score ≥ 1.5; screen potential anti-colorectal cancer drugs by matching drug target_name with the PPAR ChIP gene set and investigating the anticancer type and effect of the drug in the literature;

[0105] (3) Download the shRNA and Overexpression result tables, and obtain the corresponding gene sets according to the conditions in 4.3; perform KEGG analysis on the two gene sets and match them with the PPAR ChIP gene set KEGG results to verify that the signature genes highly represent the molecular mechanism of anthocyanin's cancer inhibition function, thereby demonstrating the effectiveness of candidate drug repositioning.

[0106] The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with data, charts, etc. of the test process.

[0107] like Figure 2 As shown in the results, the cell viability experiment showed that to a certain extent, the three anthocyanins could inhibit the proliferation of colorectal cancer cells. This is consistent with the fact that anthocyanins are small molecules against colorectal cancer in the study.

[0108] like Figure 3 As shown, the CMap matching shRNA and Overexpression gene set KEGG results showed that the functional pathway was similar to the anthocyanin functional pathway.

[0109] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for mining potential anti-colorectal cancer drugs. It is characterized in that Potential anti-colorectal cancer drugs are perampanel, fosinopril, and aminoglutethimide; Methods for discovering potential anti-colorectal cancer drugs include: By analyzing the transcriptome data of natural small molecule compounds in the treatment of colorectal cancer, we obtained the signature target gene set; then we used the CMap database to screen out the corresponding potential anti-colorectal cancer small molecule drugs; The method for mining potential anti-colorectal cancer drugs includes the following steps: Step 1: Find natural small molecule compounds that have anti-colorectal cancer effects and are related to nuclear receptors, verify the anti-cancer effects, and obtain the optimal action conditions; Step 2: Through transcriptome sequencing analysis, systematic evaluation was conducted on the effects of cyanine structure, concentration, and time on gene expression in HT29 cells; Step 3: Screening of anthocyanin characteristic gene sets and mining of potential functional pathways; Step 4: Obtain the signature gene set for CMap analysis through time series analysis and differential gene analysis results, and screen for potential colorectal cancer drugs similar to anthocyanins; In step 2, transcriptome sequencing analysis was used to systematically evaluate the effects of anthocyanin structure, concentration, and time on HT29 cell gene expression, including: (1) Transcriptome sequencing was performed on HT29 cells in the concentration, time and structure treatment groups and the control group, and the three replicate data were merged after upstream quality control; (2) Calculate the TPM value to perform cluster analysis on the full expression profile of genes under different structures, concentrations, and time; (3) rank-rank hypergeometric overlap analysis of the same / opposite expression patterns among different structures, concentrations, and times; (4) The data of the three anthocyanins at different concentrations were mean integrated and subjected to GSEA analysis to obtain the enriched functional pathways.

2. The method for mining potential anti-colorectal cancer drugs according to claim 1, It is characterized in that Other methods for discovering potential anti-colorectal cancer drugs include: Through research, we found anthocyanins, a natural small molecule compound with anti-colorectal cancer effects. After treating the HT29 colorectal cancer cell line with anthocyanins, CCK8 was used to detect cell viability to obtain the effects and optimal action conditions of anthocyanins. High-throughput data analysis was performed through transcriptome sequencing, and influencing factors were evaluated. Signature gene sets were screened out through temporal analysis and differential gene analysis, and the CMap database was used to match and screen corresponding potential anti-colorectal cancer small molecule drugs.

3. The method for mining potential anti-colorectal cancer drugs according to claim 1, It is characterized in that Step 1 is to find natural small molecule compounds with anti-colorectal cancer effects and related to nuclear receptors, verify the anti-cancer effects, and obtain the optimal action conditions including: (1) Background and literature research; Anthocyanidins are natural flavonoid compounds and have anti-colorectal cancer effects. Oxisome proliferator-activated receptors are receptors for anthocyanidins and have antioxidant lipid metabolism effects. (2) Anthocyanin treatment inhibits proliferation of colorectal cancer cell lines; Compared with the control group, the growth inhibitory effect of the treatment group first increased and then decreased with time, and increased with the increase of concentration; and the experiment proved that under the conditions of 12h and 100μM, the growth inhibitory effect was the largest, concentrations: 0.1μM, 1μM, 10μM&100μM; time: 12h, 24h, 48h&72h.

4. The method for mining potential anti-colorectal cancer drugs according to claim 3, It is characterized in that The direct nuclear receptor for anthocyanins is PPAR and the colorectal cancer cell line is HT29.

5. The method for mining potential anti-colorectal cancer drugs according to claim 1, It is characterized in that The concentrations of the treatment groups in step (1) were 1 μM and 100 μM, the treatment time was 12 h and 24 h, and the structures were no sugar group, monosaccharide group and disaccharide group.

6. The method for mining potential anti-colorectal cancer drugs according to claim 1, It is characterized in that The screening of anthocyanin characteristic gene sets and potential functional pathway mining in step 3 include: (1) Time series analysis of the three anthocyanins was performed, with 8 patterns analyzed in each group, and the pattern with significant changes within 12 h was selected; (2) The gene set with significant changes in anthocyanin 12 h was selected for KEGG pathway enrichment analysis to explore potential functional pathways; (3) By analyzing the PPARChIP dataset in existing literature, hypothesis testing and KEGG analysis were performed with overlapping gene sets to identify the key target genes of anthocyanin effects.

7. The method for mining potential anti-colorectal cancer drugs according to claim 1, It is characterized in that In step 4, the signature gene set for CMap analysis is obtained through the results of temporal analysis and differential gene analysis, and potential colorectal cancer drug screening similar to anthocyanins is performed, including: (1) The data of anthocyanin 12 h treatment were selected for differential gene analysis, and the overlap with the temporal analysis gene set was taken as the signature gene set for CMap matching analysis; (2) Perform CMap analysis on the online website CLUE; click Tools-Query, select Geneexpression, Touchstone, Latest in Query parameters; input signature gene sets according to up and down regulation, and click SUBMIT to start matching analysis; (3) Download the Compound result table and obtain a small molecule drug set by screening CRC cell lines with a norm_cs score ≥ 1.5; screen potential anti-colorectal cancer drugs by matching drug target_name with the PPARChIP gene set and literature research on drug anticancer types and effects; (4) Download the shRNA and Overexpression result tables, and obtain the corresponding gene sets according to the conditions in step (3); perform KEGG analysis on the two gene sets and match them with the PPARChIP gene set KEGG results to verify that the signature genes highly represent the molecular mechanism of anthocyanin's cancer inhibition function.

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