Medicine for preventing or treating gastric cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of hinokitiol in prevention or treatment of gastric cancer. According to the biological effect of the hinokitiol in the gastric cancer, the interaction between DNA methylated transferase DNMT1 / UHRF1 and a cancer suppressor gene ZNF154 can be regulated and controlled, so that the capacity of inhibiting proliferation and migration of the gastric cancer is achieved, and the progress of the gastric cancer is inhibited. In addition, the hinokitiol reverses the expression of the cancer suppressor gene ZNF154 in the gastric cancer cells, and cooperates with the ZNF154 to inhibit the development of the gastric cancer. The hinokitiol inhibits proliferation and migration of gastric cancer cells through multiple effects, the effect is remarkable, and the hinokitiol has guiding significance in clinic; in addition, the hinokitiol is easy to obtain and low in cost, and the prepared medicine takes effect quickly and has actual production and application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of cypermethrin in preventing / treating gastric cancer. Background Art

[0002] Gastric cancer is a common malignant tumor of the digestive tract, and its occurrence is related to multiple factors, including genetics, environment, and lifestyle. In recent years, epigenetic changes, especially DNA methylation, have played an important role in the occurrence and development of gastric cancer. DNA methylation is an important epigenetic mechanism that regulates gene expression by adding methyl groups to specific positions on DNA molecules. This mechanism can lead to the silencing of certain genes, thereby affecting the growth, differentiation, and death processes of cells. Studies have shown that there are methylation abnormalities in multiple genes in gastric cancer tissues, and these abnormalities are usually associated with the inactivation of tumor suppressor genes. Methylation modification is one of the most common epigenetic modifications and can mediate a variety of processes in cells, such as maintaining genome integrity, genomic imprinting, transcriptional regulation, and developmental processes. In cancers, including gastric cancer, global DNA hypomethylation and abnormal regional DNA hypermethylation can be observed. DNA hypermethylation has been widely studied in almost all cancers and is believed to promote cancer formation and development by inhibiting the expression of tumor suppressor genes, cell cycle regulatory genes, and DNA repair genes. DNA methylation plays an important role in the occurrence and development of gastric cancer. Analyzing the methylation status of specific genes can not only help understand the biological characteristics of gastric cancer, but also provide new targets for the early diagnosis and treatment of gastric cancer.

[0003] Abnormal DNA methylation is a hallmark of cancer, and DNMT1 (DNA methyltransferase 1) and UHRF1 (ubiquitin-like protein 1 containing PHD and RING finger domains) are key enzymes for maintaining DNA methylation. UHRF1 is overexpressed in most cancers (Xiaoying Chen, Kosuke Yamaguchi et al. "DNA methylation protects cancer cells against non-canonical senescence." bioRxiv (2024).). DNMT1 is responsible for passing the methylation pattern to the next generation during DNA replication, while UHRF1 interacts with DNMT1 through its SRA (SET and RING related) domain, helping DNMT1 to locate on hemimethylated DNA, thereby promoting DNA methylation (Ahmet C. Berkyurek, I. Suetake et al. "The DNA Methyltransferase Dnmt1 Directly Interacts with the SET and RING Finger-associated (SRA) Domain of the Multifunctional Protein Uhrf1 to Facilitate Accession of the Catalytic Center to Hemi-methylated DNA*." The Journal of Biological Chemistry (2013).; P. Bashtrykov, G. Jankevicius et al. "The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism*." The Journal of Biological Chemistry (2013).).In addition, UHRF1 also promotes the ubiquitination of histone H3 through its C-terminal ubiquitin E3 ligase activity, which also contributes to the localization and methylation activity of DNMT1 (Taosui Li, Linsheng Wang et al. "Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation." Nucleic Acids Research (2018).). The coordinated expression of UHRF1 and DNMT1 is co-regulated in different tissues and cells, and UHRF1 recruits DNMT1 in multiple ways, enhancing the activity and specificity of DNMT1.

[0004] As a transcription factor, ZNF154 is involved in a variety of biological processes, including cell proliferation, differentiation, and apoptosis. The methylation status of the ZNF154 gene has been shown to be closely related to the development, diagnosis, and prognosis of cancer in a variety of cancers. Increasing the expression of ZNF154 can inhibit the proliferation and migration of cancer cells (Wenting He, Shi-yong Lin et al. "Targeted demethylation at ZNF154 promotor upregulates ZNF154 expression and inhibits the proliferation and migration of Esophageal Squamous Carcinoma cells." Oncogene (2022).), providing new ideas for the early detection and treatment of cancer.

[0005] Hinokitiol is a compound extracted from Japanese cypress, the main components of which include α-pinene, β-pinene, etc. These components have multiple biological activities, including antibacterial, anti-inflammatory and antioxidant effects. Hinokitiol can effectively reduce the expression of proinflammatory cytokines IL-1β, IL-6 and IL-8, and reduce the accumulation of reactive oxygen species (ROS), thereby inhibiting cell apoptosis (Xu Yufeng. Study on the protective effect and mechanism of hinokitiol on human corneal epithelial cell damage induced by PolyI:C and hydrogen peroxide [D]. Zhejiang University, 2018.). Hinokitiol can also induce apoptosis of chondrosarcoma cells through the endoplasmic reticulum stress pathway, providing a new approach for cancer treatment (Fan Xiaoliang, Zhang Jian, Yu Zefeng, etc. In vitro study of hinokitiol inducing apoptosis of chondrosarcoma cells through the endoplasmic reticulum stress pathway [J]. Journal of Oncology, 2016.). However, there is no research on the relationship between hinokitiol and gastric cancer. Summary of the invention

[0006] The present invention finds that hibiscitriol inhibits the level of DNA methylation in gastric cancer cells and upregulates the expression level of ZNF154, thereby being able to effectively inhibit the proliferation and migration ability of gastric cancer cells. Based on this, the present invention is completed.

[0007] In a first aspect, the present invention provides a use of hibismol in preparing a product for inhibiting the activity of DNMT1 and / or UHRF1 methyltransferase.

[0008] Furthermore, when the activity of DNMT1 and / or UHRF1 methyltransferase is reduced, the cellular methylation level is inhibited and the occurrence of cancer is suppressed.

[0009] Furthermore, when the cells are gastric cancer cells, the activity of DNMT1 and / or UHRF1 methyltransferase is reduced, thereby reducing the methylation level of ZNF154 promoter region CpG_78~252 (Region 1) and CpG_-220~-23 (Region 2), and the proliferation and migration ability of gastric cancer cells is weakened.

[0010] Furthermore, the product that inhibits the activity of DNMT1 and / or UHRF1 methyltransferase refers to an experimental reagent or an antibacterial agent.

[0011] In a second aspect, the present invention provides a use of hibiscitol in preparing a product for upregulating the expression level of ZNF154.

[0012] Furthermore, when the expression level of ZNF154 is upregulated, the proliferation and migration ability of gastric cancer cells are weakened, and gastric cancer is inhibited.

[0013] Furthermore, the product that upregulates the expression level of ZNF154 refers to an experimental reagent or an antibacterial agent.

[0014] In a third aspect, the present invention provides a drug for preventing and / or treating gastric cancer, wherein the drug comprises hibiscus alcohol.

[0015] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating gastric cancer.

[0016] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0017] Furthermore, the drug for preventing and / or treating gastric cancer can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, lozenges, suppositories and / or lyophilized powder injections.

[0018] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0019] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0020] Furthermore, the drug for preventing and / or treating gastric cancer can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or encapsulated with other substances.

[0021] In a fourth aspect, the present invention provides a use of hibismol in the preparation of a drug for preventing and / or treating gastric cancer, wherein the drug comprises at least one of the following uses:

[0022] Inhibit DNMT1 and / or UHRF1 methyltransferase activity;

[0023] Suppress the level of DNA methylation;

[0024] Upregulate the expression level of ZNF154;

[0025] Inhibit the proliferation and migration of gastric cancer cells;

[0026] Preventing and / or treating gastric cancer.

[0027] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating gastric cancer.

[0028] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0029] Furthermore, the drug for preventing and / or treating gastric cancer can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, lozenges, suppositories and / or lyophilized powder injections.

[0030] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0031] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0032] Furthermore, the drug for preventing and / or treating gastric cancer can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or encapsulated with other substances.

[0033] Beneficial Effects

[0034] The present invention finds that the biological effect of cypermethrin in gastric cancer can achieve the ability to inhibit the proliferation and migration of gastric cancer and inhibit the progression of gastric cancer by regulating the interaction between DNA methyltransferase DNMT1 / UHRF1 and tumor suppressor gene ZNF154. In addition, cypermethrin reverses the expression of tumor suppressor gene ZNF154 in gastric cancer cells and synergistically inhibits the development of gastric cancer with ZNF154. Cypermethrin inhibits the proliferation and migration of gastric cancer cells through multiple effects, with significant effects, and has guiding significance for clinical practice; in addition, cypermethrin is easy to obtain, low in cost, and has a fast effect when made into medicine, and has practical production and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 IC50 of cypermethrin in treating gastric cancer cells.

[0036] Figure 2 Effects of different concentrations of hibiscus alcohol on gastric cancer cells.

[0037] Figure 3 Effects of different concentrations of hibiscus alcohol on ZNF154 and DNMT1 / UHRF1 methyltransferase and DNA methylation in gastric cancer.

[0038] Figure 4 Hibamol acts on gastric cancer cells overexpressing ZNF154.

[0039] Figure 5 Effect of cypermethrin on ZNF154 knockdown gastric cancer cells. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0042] Explanation of terms

[0043] Demethylation: It is a complex biological process involving multiple mechanisms and pathways. At the DNA level, demethylation refers to the process of converting methylated DNA nucleosides into unmodified nucleosides. This process plays an important role in regulating gene expression and maintaining genome stability. DNA demethylation is closely related to the occurrence and development of many diseases. For example, the high methylation state of DNA is related to the occurrence and development of tumors. Reversing the abnormal high methylation of tumor cell DNA and restoring the expression of tumor suppressor genes or DNA repair genes have become research hotspots in the development of new anti-tumor drugs.

[0044] Chromatin immunoprecipitation analysis (ChIP): The basic principle is that in the living cell state, when treated with formaldehyde, covalent bonds will be generated between proteins and proteins, proteins and nucleic acids (DNA or RNA) that are close to each other. In the cell, when TF and Promoter bind to each other, they must be close to each other or fit together. At this time, treatment with formaldehyde can generate covalent bonds between them. The fixed protein-DNA complex is randomly cut into small chromatin fragments within a certain length range by ultrasound or enzyme treatment, and then the complex is precipitated through the specific recognition reaction of antigen and antibody, specifically enriching the DNA fragments bound by the target protein, and obtaining information on the interaction between protein and DNA by purifying and detecting the target fragments. Screen unknown DNA information that interacts with the target protein through qPCR or second-generation sequencing.

[0045] Methylated DNA immunoprecipitation sequencing technology (MeDIP Sequencing): The genome is broken into small fragments by ultrasound, and the highly methylated fragments are enriched using antibodies against 5′-methylcytosine. MeDIP combined with high-throughput sequencing technology can be used to enrich the DNA methylated regions in the genome and then perform high-throughput sequencing. It can quickly and effectively find the methylated regions on the genome and perform whole-genome DNA analysis between multiple samples, thereby comparing the differences in DNA methylation modification patterns between different cells, tissues, and even disease samples.

[0046] MTT test: MTT is a yellow or orange powdered chemical reagent, with the Chinese name Thiazole Blue. Its main function is to detect cell survival and growth. Therefore, its uses mainly include two aspects: 1. Detection of the cytotoxicity of drugs or other intervention methods on cells cultured in vitro; 2. Cell proliferation and activity determination. The detection principle of MTT is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function.

[0047] Transwell experiment: Its basic principle is to put a small chamber into a culture plate. The small chamber is called the upper chamber, and the culture plate is called the lower chamber. The upper and lower culture fluids are separated by a polycarbonate membrane. The upper culture fluid is added to the upper chamber, and the lower culture fluid is added to the lower chamber. The cells to be studied are planted in the upper chamber. Since the polycarbonate membrane is permeable, the components in the lower culture fluid can affect the growth and movement of the cells in the upper chamber.

[0048] Experimental Materials

[0049] Hinokitiol, molecular formula: C 10 H 12 O2, the structural formula is as follows:

[0050]

[0051] Gastric cancer AGS cell line: originated from the Cell Bank of the Chinese Academy of Sciences.

[0052] Example 1 IC50 of cypermethrin on gastric cancer cells

[0053] A. Test Methods

[0054] (1) Prepare different concentrations of cypermethrin solutions using complete culture medium: 0 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, and 1000 μM;

[0055] (2) digesting gastric cancer cells AGS in the logarithmic growth phase, centrifuging and resuspending;

[0056] (3) The cell suspension in (2) was adjusted to 1.5×10 3 Cells / well were inoculated into the well plate, and the AGS cells were cultured with the complete medium prepared in (1) containing different concentrations of hibismol;

[0057] (4) After 24 h, the cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit;

[0058] (5) Detect the absorbance at 490 nm using an enzyme-labeled instrument.

[0059] B. Test Results

[0060] like Figure 1 As shown, the IC50 of hibismol in gastric cancer cells is 20.29 μM.

[0061] Example 2 Optimal concentration of cypermethrin for the action of gastric cancer cell AGS

[0062] A. Effect of different concentrations of hibismol on gastric cancer cells AGS

[0063] (1) Prepare different concentrations of cypermethrin solutions using complete culture medium: 5 μM, 10 μM, and 20 μM;

[0064] (2) digesting gastric cancer cells AGS in the logarithmic growth phase, centrifuging and resuspending;

[0065] (3) treated with different concentrations of cypermethrin prepared in (1);

[0066] (4) digesting the solution in (3) with trypsin, centrifuging, removing the supernatant, and collecting the cell precipitate;

[0067] (5) Lysing the cells collected in (4) with TRIzol reagent and further extracting RNA;

[0068] (6) using a reverse transcription kit to reverse transcribe the RNA extracted in (5) into cDNA;

[0069] (7) The cDNA obtained in (6) was amplified using a qPCR kit to further analyze the mRNA expression level of ZNF154.

[0070] B. MTT assay

[0071] (1) Prepare different concentrations of cypermethrin solutions using complete culture medium: 5 μM, 10 μM, and 20 μM;

[0072] (2) digesting gastric cancer cells AGS in the logarithmic growth phase, centrifuging and resuspending;

[0073] (6) The cell suspension in (2) was adjusted to 1.5×10 3 Cells / well were inoculated into the well plate, and the AGS cells were cultured with the complete medium prepared in (1) containing different concentrations of hibismol;

[0074] (3) Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) detection kit at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h;

[0075] (4) Detect the absorbance at 490 nm using an enzyme-labeled instrument.

[0076] C. Transwell experiment

[0077] (1) Prepare different concentrations of cypermethrin solutions using complete culture medium: 5 μM, 10 μM, and 20 μM;

[0078] (2) digesting gastric cancer cells AGS in the logarithmic growth phase, centrifuging and resuspending;

[0079] (3) The cell suspension in (2) was adjusted to 2×10 4 The cells were seeded in the chamber of the Tranwell plate, and the AGS cells were cultured with the complete medium prepared in (1) containing different concentrations of hibismol;

[0080] (4) After 24 hours, the cells treated in (3) were fixed with paraformaldehyde, washed, and stained with crystal violet to observe cell migration.

[0081] D. Test results

[0082] like Figure 2 As shown in Figure 2, different concentrations of cypermethrin acted on gastric cancer cells and the expression level of ZNF154 was upregulated. Among them, C1: 5μM, C2: 10μM, C3: 20μM. When the concentration was 10μM, cypermethrin exerted its effect thoroughly (as shown in Figure 2). Figure 2 Therefore, a concentration of 10 μM was selected for subsequent experiments.

[0083] like Figure 2 As shown in B, when cypermethrin was added, the proliferation ability of gastric cancer cells decreased with the increase of concentration.

[0084] like Figure 2 As shown in C, through cell staining, compared with the control group, the proliferation and migration abilities of gastric cancer cells were decreased after being treated with hibiscus alcohol; and as the concentration of hibiscus alcohol solution treatment increased, the proliferation and migration abilities of gastric cancer cells decreased.

[0085] Example 3 Effect of cypermethrin on methyltransferase and DNA methylation levels in gastric cancer cells

[0086] A. Chromatin Immunoprecipitation Assay (CHIP)

[0087] (1) Prepare different concentrations of cypermethrin solutions using complete culture medium: 5 μM, 10 μM, and 20 μM;

[0088] (2) digesting gastric cancer cells AGS in the logarithmic growth phase, centrifuging and resuspending;

[0089] (3) digesting the solution in (2) with trypsin, centrifuging, removing the supernatant, and collecting the cell precipitate;

[0090] (4) Extracting DNA from cells;

[0091] (5) Combine the DNA in (4) with 2 μg of DNMT1 and UHRF1 antibodies overnight;

[0092] (6) The DNA fragments released by co-immunoprecipitation were digested with proteinase K and then purified using a QIAquick purification kit;

[0093] (7) Use qPCR to detect the enrichment of the DNA precipitated in (6).

[0094] B. Methylated DNA immunoprecipitation (MeDIP)

[0095] 1) Prepare different concentrations of cypermethrin solutions using complete culture medium: 5 μM, 10 μM and 20 μM;

[0096] 2) digesting gastric cancer cells AGS in the logarithmic growth phase, centrifuging and resuspending;

[0097] 3) digesting the solution in (2) with trypsin, centrifuging, removing the supernatant, and collecting the cell precipitate;

[0098] 4) Extract DNA from cells;

[0099] 5) Combine the DNA in (4) with anti-5mC or nonspecific IgG overnight;

[0100] 6) The DNA fragments released by co-immunoprecipitation were digested with proteinase K and then purified using QIAquick purification kit;

[0101] 7) Use qPCR to detect the enrichment of the DNA precipitated in (6).

[0102] C. Test Results

[0103] like Figure 3 As shown in A, in gastric cancer cells treated with hibiscus alcohol, the amount of ZNF154 DNA (CpG_78~252 (Region 1) and CpG_-220~-23 (Region 2)) bound to DNMT1 / UHRF1 decreased, which was consistent with Figure 2 As shown in Figure 2, the induced ZNF154 expression was increased ( Figure 2 ZNF154 was elevated).

[0104] like Figure 3 As shown in B, after the gastric cancer cells were treated with hibiscitriol, the methylation levels of ZNF154 CpG_78~252 (Region 1) and CpG_-220~-23 (Region 2) fragments decreased, indicating that hibiscitriol inhibited the methylation of ZNF154 CpG_78~252 (Region 1) and CpG_-220~-23 (Region 2) associated with UHRF1 / DNMT1.

[0105] Example 4 Effect of cypermethrin on gastric cancer cells with overexpression of ZNF154

[0106] A. Construction of ZNF154 overexpressing gastric cancer cells

[0107] (1) Digest gastric cancer cells, resuspend and count, inoculate into well plates, and wait for them to adhere to the wall;

[0108] (2) Gastric cancer cells were infected with ZNF154-overexpressing lentivirus and screened with puromycin to obtain a gastric cancer cell line that stably overexpressed ZNF154.

[0109] B. Effect of hibismol on ZNF154 mRNA level in ZNF154 overexpressing gastric cancer cells

[0110] (1) ZNF154-overexpressing gastric cancer cells were digested, centrifuged, resuspended and counted;

[0111] (2) using complete culture medium to prepare a 10 μM hibismol solution, and using it to culture the cells in (1);

[0112] (3) digesting the solution in (2) with trypsin, centrifuging, removing the supernatant, and collecting the cell precipitate;

[0113] (4) Lysing the cells in (3) using TRIzol reagent, and then extracting RNA;

[0114] (5) using a reverse transcription kit to reverse transcribe the RNA obtained in (4) into cDNA;

[0115] (6) The cDNA obtained in (5) was amplified using a qPCR kit to further analyze the mRNA expression level of ZNF154.

[0116] C. MTT test

[0117] (1) Gastric cancer cells overexpressing ZNF154 were digested with trypsin and resuspended by centrifugation;

[0118] (2) The cell suspension was adjusted to 1.5×10 3 Cells / well were seeded into the well plate;

[0119] (3) Prepare a 10 μM hibismol solution using complete culture medium and use it to culture the cells obtained in (2). The treatment time is: 0,

[0120] 24,48,72,96,120 hours;

[0121] (4) Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit;

[0122] (5) Detect the absorbance at 490 nm using an enzyme-labeled instrument.

[0123] D. Transwell experiment

[0124] (1) ZNF154-overexpressing gastric cancer cells were digested with trypsin, centrifuged, resuspended, and counted;

[0125] (2) The cell suspension was adjusted to 2×10 4 The cells were seeded in the chambers of Tranwell plates;

[0126] (3) preparing a 10 μM hibismol solution using complete medium, and culturing the cells obtained in (2) with the solution;

[0127] (4) Fix the cells treated in (3) with paraformaldehyde, wash them, stain them with crystal violet, and observe the migration of the cells.

[0128] E. Test results

[0129] like Figure 4 As shown in A, hibismol can further upregulate the expression of ZNF154 in ZNF154-overexpressing cells ( Figure 4 A); Figure 4 As shown in B and 4C, the proliferation and migration abilities of gastric cancer cells were synergistically inhibited.

[0130] Example 5 Effect of cypermethrin on ZNF154 knockdown gastric cancer cells

[0131] A. Construction of ZNF154 knockdown gastric cancer cell line

[0132] (1) Digest gastric cancer cells, resuspend and count, inoculate into well plates, and wait for them to adhere to the wall.

[0133] (2) Gastric cancer cells were infected with ZNF154 knockdown lentivirus and screened with puromycin to obtain a gastric cancer cell line with stable ZNF154 knockdown.

[0134] B. Effect of Hibamol on ZNF154 Knockdown Gastric Cancer Cells

[0135] (1) A 10 μM hibismol solution was prepared using complete culture medium, and the ZNF154 knockdown gastric cancer cells were cultured therewith, followed by trypsin digestion, centrifugation, removal of the supernatant, and collection of the cell pellet;

[0136] (2) Lysing the cells treated in (1) with TRIzol reagent and then extracting RNA;

[0137] (3) reverse transcribe RNA into cDNA using a reverse transcription kit;

[0138] (4) cDNA was amplified using a qPCR kit to further analyze the mRNA expression level of ZNF154.

[0139] C. MTT assay

[0140] (1) Gastric cancer cells with ZNF154 knockdown were digested with trypsin and resuspended by centrifugation;

[0141] (2) The cell suspension in (1) was adjusted to 1.5×10 3 Cells / well were seeded into the well plate;

[0142] (3) Prepare a 10 μM hibismol solution using complete culture medium and use it to culture the cells obtained in (2). The treatment time is: 0,

[0143] 24,48,72,96,120 hours;

[0144] (4) Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit;

[0145] (5) Detect the absorbance at 490 nm using an enzyme-labeled instrument.

[0146] D. Transwell experiment

[0147] (1) Gastric cancer cells with ZNF154 knockdown were digested with trypsin, centrifuged, resuspended, and counted;

[0148] (2) The cell suspension in (1) was adjusted to 2×10 4 The cells were seeded in the chambers of Tranwell plates;

[0149] (3) preparing a 10 μM hibismol solution using complete medium, and culturing the cells obtained in (2) with the solution;

[0150] (4) Fix the cells treated in (3) with paraformaldehyde, wash them, stain them with crystal violet, and observe the migration of the cells.

[0151] E. Test results

[0152] like Figure 5 As shown, cypermethrin had no effect on the expression level of ZNF154 in ZNF154 knockdown cells ( Figure 5 A), but can further synergistically inhibit the proliferation and migration ability of gastric cancer cells ( Figure 5 B, C). These results suggest that ZNF154 is a key downstream molecule of hibiscus alcohol in inhibiting gastric cancer.

Claims

1. A use of hibismol in the preparation of a product for inhibiting the activity of DNMT1 and / or UHRF1 methyltransferase; the product for inhibiting the activity of DNMT1 and / or UHRF1 methyltransferase refers to an experimental reagent or an antibacterial agent.

2. The DNMT1 and / or UHRF1 methyltransferase activity as described in claim 1, when the DNMT1 and / or UHRF1 methyltransferase activity in gastric cancer cells is reduced, the methylation level of the ZNF154 promoter region CpG_78~252 (Region 1) and CpG_-220~-23 (Region 2)) is reduced, and the proliferation and migration ability of gastric cancer cells is weakened.

3. A use of hibismol in the preparation of a product for upregulating the expression level of ZNF154; the product for upregulating the expression level of ZNF154 refers to an experimental reagent or an antibacterial agent.

4. By upregulating the expression level of ZNF154 as described in claim 3, the proliferation and migration abilities of gastric cancer cells are weakened, and gastric cancer is inhibited.

5. A drug for preventing and / or treating gastric cancer, comprising hibismol.

6. Use of hibismol in the preparation of a drug for preventing and / or treating gastric cancer, wherein the drug comprises at least one of the following uses: Inhibit DNMT1 and / or UHRF1 methyltransferase activity; Suppress the level of DNA methylation; Upregulate the expression level of ZNF154; Inhibit the proliferation and migration of gastric cancer cells; Preventing and / or treating gastric cancer.

7. One or more pharmaceutically acceptable carriers or excipients may also be added to the drug for preventing and / or treating gastric cancer as claimed in any one of claims 5 or 6.

8. The carrier material as claimed in claim 7 includes but is not limited to one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

9. The drug for preventing and / or treating gastric cancer as described in any one of claims 5 or 6 can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

10. The drug for preventing and / or treating gastric cancer as claimed in any one of claims 5 or 6 can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or encapsulated with other substances.

Images