| Objective:Gastric cancer,as one of the most common malignant tumors in the digestive tract,poses a serious threat to human health.Although the incidence rate and mortality of gastric cancer in China are declining,the burden of the disease is still heavy.Zuojinwan is a traditional Chinese medicine formula in China.Since its establishment,it has been widely used in the treatment of gastric diseases.In recent years,its compound and monomers have developed rapidly in clinical application and experimental research in the prevention and treatment of gastric cancer.With the birth of network pharmacology,due to its holistic and systematic characteristics,as well as the"holistic view"characteristics of traditional Chinese medicine and the"multi component,multi pathway,and multi target"characteristics of traditional Chinese medicine prescriptions,it shares similarities and differences.As an important tool for the modernization of traditional Chinese medicine research,it has achieved outstanding results in the development of new Chinese medicine,research on traditional Chinese medicine syndromes,adverse reactions and toxic side effects of traditional Chinese medicine,and other aspects,Especially in explaining the compatibility laws of traditional Chinese medicine formulas,predicting the target and signaling pathways of traditional Chinese medicine formulas,analyzing the active ingredients and mechanisms of action of traditional Chinese medicine formulas,and so on.Micro RNAs are a type of noncoding small RNAs with a length of 20-21 nt that can negatively regulate gene expression after transcription by specifically binding to corresponding m RNA.More than a thousand miRNAs have been discovered,of which miR-107 is associated with the occurrence,development,invasion,and metastasis of various malignant tumors,including gastric cancer.Given the differential expression in gastric cancer tissue and its regulatory role in the occurrence and development of gastric cancer,miR-107 may serve as a biomarker for the diagnosis,treatment,and prognosis of gastric cancer.Its expression imbalance is consistent with the pathogenesis of traditional Chinese medicine’s"deficiency,evil,and excess"in gastric cancer.On the basis of network pharmacology and miR-107,this study explores the anti-gastric cancer effects and possible mechanisms of Zuojinwan from three levels:molecular,cellular,and animal,through theoretical,clinical,and experimental research,providing theoretical and experimental basis for Zuojin wan’s treatment of gastric cancer..Methods:1.Network pharmacology:Retrieve the effective active ingredients and corresponding targets of Zuojinwan on the TCMSP platform,use Genecards,OMIM,Dis Ge NET,and TTD databases to obtain disease targets related to gastric cancer,and predict the target genes of miR-107 on Target Scan,miRDB,miRWalk,and miRBase databases.After screening the common targets of Zuojinwan,miR-107,and gastric cancer,a PPI network was constructed using STRING database.Cytoscape software was used for visualization analysis,and David database was used for GO enrichment analysis and KEGG enrichment analysis of the obtained results.2.Clinical research section:Develop inclusion and exclusion criteria for gastric cancer patients,record general information such as age,gender,tumor diameter,tumor markers（CEA,CA19-9）,degree of differentiation,lymph node metastasis,infiltration,distant metastasis,and TNM staging of the included patients.Collect gastric cancer tissues and paracancerous tissues from patients with gastric cancer who underwent surgical resection.Real time PCR was used to detect the level of miR-107 in gastric cancer tissues and paracancerous tissues.Western blot was used to detect the level of RUNX1T1in gastric cancer tissues and paracancerous tissues.The binding sites of miR-107 and RUNX1T1 were predicted through Target Scan online database.The target relationship between miR-107 and RUNX1T1 was verified through double Luciferase reporter gene experiment.3.Cell experiment research section:Using CCK-8 method to detect the inhibitory effect of different concentrations of Zuojin Wan containing serum on the growth of gastric cancer MGC-803 cells,and determine the low,medium,and high doses of the test drug in the cell experiment;Detect the inhibitory effect of drugs on cell proliferation through cell cloning experiments;Detect the inducing effect of drugs on cell apoptosis through AO/EB double staining experiment and flow cytometry;Detect the effect of drugs on cell cycle through flow cytometry;The effect of drugs on cell migration was detected by scratch healing test and Transwell test;Western blot was used to detect changes in the expression of related proteins in cells treated with drugs.4.Animal experimental research part:A subcutaneous transplanted tumor nude mouse model was established by subcutaneous injection of gastric cancer MGC-803 cell suspension.The growth status of the transplanted tumor was observed after treatment with Zuojinwan decoction by gavage,and the pathological changes of the transplanted tumor tissue were observed and analyzed using tissue sectioning HE staining method;Immunohistochemical method was used to detect the expression of RUNX1T1;Real time PCR method was used to detect the expression level of miR-107.Results:1.33 active ingredients of Zuojinwan were identified,with 209 corresponding targets,12169 targets for gastric cancer related diseases,and 14878 target genes for miR-107;We screened 147 potential targets for Zuojinwan to treat gastric cancer through miR-107.Visualization analysis to obtain core effective components such as quercetinβ-Glutinosterol and other core targets such as AKT1 and TP53 are enriched in the cancer pathway and PI3K-AKT signaling pathway.2.Compared with adjacent tissues,the average expression level of miR-107 in gastric cancer tissue is higher,while the relative expression level of RUNX1T1 is lower（P<0.05）;Moreover,the expression of miR-107 and RUNX1T1 in gastric cancer is correlated with tumor differentiation,lymph node metastasis,TNM staging,and depth of invasion（P<0.05）,but not with patient gender,age,tumor size,and lesion location（P>0.05）.The expression of miR-107 and RUNX1T1 in gastric cancer tissue was negatively correlated.It was verified that RUNX1T1 was the target gene of miR-107 by double Luciferase reporter gene experiment.3.There is a time concentration dependent effect(*P<0.05 or**P<0.01 or***P<0.001)on the inhibition of cell growth of Zuojinwan medicated serum.After 48 hours of IC50,5%,10%,and 20%were determined as low,medium,and high Zuojinwan medicated serum concentrations for subsequent experiments.Compared with the drug-free serum group,the cell clone size and number in each Zuojinwan containing serum group（5%group:23.26%±1.53;10%group:49.37%±0.88;20%group:64.35%±0.91）decreased（*P<0.05）;The proportion of S phase cells significantly increased（5%group:24.55%±0.89,10%group:29.33%±0.99,20%group:32.90%±1.05,*P<0.05）;Significantly induce apoptosis in MGC-803 cells,with an apoptosis rate of 21.58%±1.39 in the 5%group,32.17%±1.50 in the10%group,and 43.66%±1.95 in the 20%group;The cell migration rate was significantly reduced(*P<0.05 or**P<0.01 or***P<0.001).The migration rate of scratch test was 35.78%±2.09 in the 5%group,24.83%±2.38 in the 10%group,and 5.69%±3.11 in the 20%group.The migration rate of Transwell test was 42.12%±1.23 in the 5%group,59.03%±1.50 in the 10%group,and77.28%±1.95 in the 20%group.After treatment with Zuojinwan containing serum,the expression of E-cadherin,Cyclin E,Bax,Fas,FADD,Caspase-8,and Caspase-3 in gastric cancer MGC-803 cells was significantly upregulated,while the expression of Cyclin A,Cyclin B1,Bcl-2,p PI3K,p AKT,and pm TOR was significantly downregulated（*P<0.05）.4.After being treated with Zuojinwan by gavage in nude mice bearing gastric cancer,compared with the control group,the volume of transplanted gastric cancer MGC-803 cells decreased,the mass decreased,and the tumor growth inhibition rate was 45.83%（*P<0.05）.HE staining analysis revealed that the tumor cells in the Zuojinwan group exhibited degenerative changes,nuclear pyknosis,and fibrosis,while the cell structure in solid tumors showed necrotic areas of nuclear loss and fragmentation;Immunohistochemical and ENCE results showed high expression of RUNX1T1;The Real time PCR method showed a significant decrease in miR-107 levels（*P<0.05）.Conclusion:1.The process of Zuojinwan treating gastric cancer through miR-107 is the overall regulation of multiple targets,multiple pathways,multi-dimensional regulation,and synergy.The specific mechanism may be that Zuojinwan interferes with the process of RNA polymerase II promoter transcription,transcription,DNA templating,drug response,and apoptosis during the occurrence and development of gastric cancer through cancer pathways,P13K/AKT/m TOR and other signal pathways.2.The high expression of miR-107 and low expression of RUNX1T1 in gastric cancer tissue serve as potential new targets for miR-107.Inhibiting them can promote RUNX1T1 expression,and their abnormal expression is closely related to tumor differentiation,lymph node metastasis,TNM staging,and depth of invasion.They may serve as molecular markers for the diagnosis,treatment,and prognosis evaluation of gastric cancer patients.3.Zuojinwan exerts its anti-gastric cancer effect by activating the miR-107/RUNX1T1 axis.Its mechanism of action may be to inhibit cell proliferation by inhibiting the activation of the PI3K/AKT/m TOR pathway,induce cell cycle S-phase arrest by regulating the expression of Cyclin A,Cyclin B1,and Cyclin E,inhibit migration by increasing E-cadherin expression,and induce cell apoptosis through the Fas death receptor pathway and Bcl-2/Bax pathway. |
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