| Background:Investigations have shown that Helicobacter pylori is a major risk factor for gastric cancer,and about 90% of new cases of non-cardiac cancer are associated with Helicobacter pylori infection.As one of the main pathogenic factors of Helicobacter pylori,cytotoxin-related gene A(CagA)protein is encoded by the cagA gene on the cag pathogenic island(cag PAI)of Helicobacter pylori.CagA protein is secreted into the host's gastric mucosal epidermal cytoplasm via type IV secretion system,and then tyrosine phosphorylation and specific binding to the SH2 domain of SHP2 form the CagA-SHP2 complex.CagA-SHP2 complex can induce cells to elongate to form unique "hummingbird-like" changes,which can induce the occurrence of gastric cancer and promote the development of gastric cancer.Salvia miltiorrhiza is a traditional herbal medicine that is included in the Chinese Pharmacopoeia and is widely used in the field of traditional Chinese medicine.Because of its anti-inflammatory,anti-oxidant,anti-coagulant and anti-cancer effects,salvia miltiorrhiza has been used in the treatment of cardiovascular diseases and various cancers in recent years.As a natural compound extracted from the roots and rhizomes of salvia miltiorrhizae,cryptotanshinone has been found to be useful in the treatment of lung cancer,breast cancer,ovarian cancer,osteosarcoma and other cancers.It has been found that cryptotanshinone and its analog iso-tanshinone can inhibit the growth and migration of gastric cancer cells,and whether there is an intervention effecton the enhancement of gastric cancer cell proliferation and migration ability caused by CagA protein has not been studied.Therefore,we explored and verified the potential and molecular mechanism of cryptotanshinone in the treatment of gastric cancer caused by H.pylori CagA-positive strains through network pharmacological methods and experiments.ObjectiveIt has been found that cryptotanshinone and its analog iso-tanshinone can inhibit the growth and migration of gastric cancer cells,and whether there is an intervention effect on the enhancement of gastric cancer cell proliferation and migration ability caused by CagA protein has not been studied.Therefore,we explored and verified the potential and molecular mechanism of cryptotanshinone in the treatment of gastric cancer caused by H.pylori CagA-positive strains through network pharmacological methods and experiments.Methods1.Target genes for cryptotanshinone and Helicobacter pylori cytotoxicity-related gene A(CagA)proteina)Obtaining the target gene of cryptotanshinone: Obtain the target gene of cryptotanshinone by entering the Swiss Target Prediction(http://www.swisstargetprediction.ch/)database.b)Obtain target genes of CagA protein from multiple databases: through the Human Gene Database(Gene Cards;http://www.genecards.org/)and the Mendelian OnlineGenetic Database(OMIM: https: //www.ncbi.nlm.nih.gov / omim)to obtain CagA target genes.2.Construction of co-expressed target genes and gene network mapsa)Preliminary screening of target genes: The String database(https://string-db.org)is used to evaluate protein interactions between cryptotanshinone and CagA targets.Only protein interactions with a confidence level greater than 0.900 are considered.b)Constructing a network diagram of target genes: input the target gene data obtained through preliminary screening into the network visualization software Cytoscape(http://cytoscape.org/,version 3.7.1),and use the software to construct drug target genes Network diagram and disease target gene network diagram.c)Constructing a Venn diagram to obtain co-expressed target genes for drugs and diseases: identifying the hidden genes by the Venn diagram(Venny,https://bioinfogp.cnb.csic.es/tools/venny/index.html,version 2.1)The common genetic target of tanshinone and CagA is a potential target for cryptotanshinone in the treatment of gastric cancer caused by CagA-positive H.pylori strains.3.Molecular docking simulationSYBYL-X molecular docking simulation software was used to simulate the 3D structure of the protein encoded by the screened gene and cryptotanshinone.4.Transfect plasmids to construct AGS cells expressing CagA proteinThe same amount of p SR? and p SR?-HA-CagA plasmids were transfected into gastric cancer cell line AGS cells,and the expression level of CagA in the cells was detected by Western blot.5.Proliferation of human gastric cancer AGS cells after transfection and drug treatmentAGS cells transfected with plasmid were treated with cryptotanshinone at different concentrations,and the proliferation ability of AGS cells was detected using the CCK-8 kit.6.Migration and invasion of human gastric cancer AGS cells after transfection and drug treatmentTransfection of AGS cells with plasmid was treated with cryptotanshinone at different concentrations,and the migration and invasion ability of AGS cells after drug treatment were detected with the Transwell chamber.7.Changes in protein expression levels in AGS cells after transfection and drug treatmentThe transfection AGS cells were treated with cryptotanshinone at different concentrations,and the protein expression levels in transfected AGS cells after cryptotanshinone treatment were detected by Western blot after protein extraction.Results1.Obtain 100 target genes of cryptotanshinone and 370 target genes of CagA from the database.2.Nine genes co-expressed by cryptotanshinone and CagA were initially obtained:STAT3,PTPN1,PTPN6,PTPN11,MAPK10,PTK2,PTGS2,EGFR and NOS2 as potential targets for cryptotanshinone in the treatment of gastric cancer caused by CagA.3.Four genes were further screened by molecular simulation: proteins encoded by NOS2,PTK2,PTPN11 and MAPK10 have higher binding potential to cryptotanshinone.4.CagA transfection into AGS cells can cause cell proliferation,migration and invasion.5.After treatment with cryptotanshinone,the proliferation,migration and invasion ability of AGS cells expressing CagA protein were inhibited.6.CagA can cause phosphorylation of SHP2 protein in AGS cells,and cryptotanshinone can reduce the expression levels of SHP2 and p-SHP2 protein.ConclusionThe expression of Helicobacter pylori's cytotoxicity-related gene A(CagA)protein can enhance cell proliferation,migration and invasion ability.Cryptanthinone can inhibit cell proliferation,migration and invasion ability by reducing the expression of SHP2 protein and phosphorylated SHP2 protein. |
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