Study On The Inhibitory Effect And Mechanism Of Dioscin Targeted Cx43 On Gastric

Gastric cancer(GC),one of the most common types of cancer worldwide,is a highly aggressive and heterogeneous malignancy and is the fifth leading cause of cancer death worldwide.Although gastric cancer cases have declined in many regions over the past few decades,it remains a significant health problem in some areas,particularly Asia and Latin America.Gastric cancer usually has no apparent symptoms in the early stage,so it is difficult to be detected and treated at an early stage.Currently,the clinical treatment for gastric cancer is based on surgery,radiotherapy and targeted therapy,but the morbidity and mortality rate of gastric cancer remains high due to its highly invasive and metastatic biology.Therefore,we need to further investigate the pathogenesis of gastric cancer and the biomarkers that predict the prognosis of gastric cancer and find a drug that can inhibit the invasion and metastasis.Connexin 43 is a type of intercellular junction protein which is mainly expressed in cardiomyocytes,vascular endothelial cells,neuronal cells and various cancer cells.Its main function is to form an intercellular junction(gap junction),which transmits protons,charged substances,small molecule ions and signal molecules between cells,thus maintaining intracellular homeostasis,mutual coordination and mutual coordination.In addition,Cx43 protein also involves in various physiological and pathological processes such as apoptosis,cell differentiation,cell cycle regulation,cell migration and tumor metastasis.In recent years,many studies have shown that abnormal expression or mutation of Cx43 gene is associated with various diseases,especially in cancer treatment,which has attracted widespread attention.Some studies have found that some drugs can inhibit tumor growth and metastasis by regulating Cx43 gene expression and function,thus becoming a potential therapeutic strategy.However,the expression and related mechanisms of action in gastric cancer have not been investigated and deserve further exploration.Traditional Chinese Medicine(TCM)has been practiced for thousands of years and is now widely accepted as an alternative treatment for cancer.Dioscorea cirrhosa Lour,also known as Huai yam,is a vine of the dioscoreaceae family with a sweet and flat taste,and has the ability to invigorate spleen-stomach and replenish Qi,strengthen the spleen and stomach,nourish Yin and moisten the dryness,etc.It can also stimulate and regulate the human immune system,and has anti-tumor,anti-virus,and anti-aging effects.Dioscin is the most important class of compounds in dioscorea opposita,which has a variety of bioactive effects such as antioxidant,anti-inflammatory and anti-tumor.Previous studies have reported that dioscin has potent anticancer effects on various cancer cells,including gastric cancer,colon cancer,melanoma,breast cancer,endometrial cancer and leukemia,but the specific mechanism of action is still not very clear and needs further study.The previous study of our group has confirmed that dioscin has noticeable anti-tumor effects and is effective in inhibiting tumor cell proliferation,migration and invasion.Therefore,this thesis’s main objective is to investigate dioscin’s role and molecular mechanism of dioscin in inhibiting epithelial-mesenchymal transformation in gastric cancer based on network pharmacology and biochemical analysis.This study is divided into four main parts.Part Ⅰ Study of the core target genes and signaling pathways of dioscin against gastric cancer based on network pharmacologyObjective:To predict the material basis and mechanism of action of dioscin,the active ingredient of dioscorea opposita,against gastric cancer by using network pharmacology and molecular docking techniques.Methods:The Swiss Target Prediction,OMIM and Genecards databases are firstly used to retrieve the potential targets of action of dioscin and gastric cancer-related genes,respectively.Then the Venny 2.1.0 online tool is used to obtain the Venn diagrams of key targets of dioscin against gastric cancer.Subsequently,the STRING database and Cytoscape software is used to analyze protein interactions and establish PPI models.Then the material basis and mechanism of action of dioscin against gastric cancer are investigated by GO enrichment analysis and KEGG pathway enrichment analysis.And finally,molecular docking is completed by Auto Dock Tools software for validation.Results:There are 47 core target genes of dioscin against gastric cancer.Enrichment analysis show that PI3K-Akt signaling pathway is a vital target pathway,where two core target genes,AKT1 and MTOR,are on this signaling pathway.Molecular docking results show good binding activity between dioscin and AKT1 and MTOR.Conclusion:Dioscin may exert anti-gastric cancer effects by acting on AKT1 and MTOR and through PI3K-Akt signaling pathway.Part Ⅱ Study the correlation between GJA1(Cx43)gene expression and gastric cancer based on raw signal analysisObjective:To explore the potential mechanism of GJA1(Cx43)in gastric cancer and its immune target therapy by raw signal analysis.Methods:RNA sequencing data and clinical characterization data of gastric cancer are downloaded with the help of TCGA database,and the limma,survival,survminer,ggpubr and ComplexHeatmap packages are used to reflect the GJA1 expression levels and the relationship with clinical parameters and patient survival prognosis.The correlation between GJA1 expression and clinical characteristics is explored by univariate and multifactorial analyses.The differential genes in the high and low risk groups of GJA1 in gastric cancer are subjected to GO enrichment analysis and KEGG pathway enrichment analysis.Results:GJA1 may be an independent prognostic indicator for prognostic risk models,and a tabulated plot of this risk score predicts the overall survival time of patients with gastric cancer.By enrichment analysis of KEGG pathways,the top ranked pathways are PI3K-Akt signaling pathway,calcium signaling pathway,cAMP signaling pathway and cGMP-PKG signaling pathway.Conclusion:GJA1 may be a good prognostic marker and can inhibit the development of gastric cancer by regulating the PI3K-Akt signaling pathway.Part Ⅲ Effect and mechanism of diosgenin targeted connexin 43 on gastric cancer cells Objective:To study the inhibitory effect of diosgenin on proliferation,migration and invasion of gastric cancer cells and the mechanism of targeting connexin 43 to regulate PI3K/Akt/mTOR signal pathway.Methods:The mutant Cx43G21R stable cell line was constructed by lentivirus infection.The protein expression level of Cx43 was detected by Western blot.The gap junctional communication was detected by scratch labeling fluorescent dye tracer technique.The effects of mutant Cx43G21R and dioscin on cell proliferation were detected by MTT.Scratch test and transwell invasion and migration test were used to explore the effects of mutant Cx43G21R and dioscin on cell migration and invasion.Western Blot was used to detect the expression of Cx43,PI3K/Akt/mTOR signaling pathway and EMT-related proteins in human gastric cancer AGS cells treated with mutant Cx43 gene and different concentrations of dioscin for 48 h.Results:The expression level of Cx43 protein in mutant Cx43G21R increased and the ability of fluorescence transmission decreased significantly.After administration of dioscin,the ability of fluorescence transmission was restored.Compared with the control group,the proliferation activity,migration and invasion ability of mutant Cx43G21R increased significantly,and decreased significantly after the intervention of dioscin.The results of Western Blot showed that the expression of Cx43 protein in mutant Cx43G21R increased.After administration,the expression of Cx43 protein further increased in a concentration-dependent manner.Compared with the control group,the expression of p-PI3K,p-Akt and p-mTOR protein of mutant Cx43G21R increased,and decreased significantly after the intervention of dioscin,but the expression level of PI3K,Akt and mTOR protein had no significant difference.The expression level of E-cadherin protein increased,while the expression level of N-cadherin,Vimentin and Snail protein decreased.Conclusion:Dioscin can up-regulate Cx43-mediated gap junction communication and regulate mTOR/PI3K/Akt signal pathway,thus inhibiting the proliferation,migration and invasion of gastric cancer cells.Part Ⅳ Study on the effect and mechanism of diosgenin targeting connexin 43 on gastric cancer in vivoObjective:To study the effect and mechanism of diosgenin on proliferation and epithelialmesenchymal transformation of gastric cancer in vivo.Methods:The model of subcutaneous transplanted tumor in nude mice was established.The nude mice were randomly divided into four groups:Ctrl group,Cx43G21R group and dioscin group(Cx43G21R+30 mg/kg,Cx43G21R+60 mg/kg).The body weight and the size of the transplanted tumor were measured every three days.After 24 days of administration,the tumor was removed,and then the expression of related proteins in tumor tissue was detected by immunohistochemistry and Western Blot.Results:Compared with Ctrl group,the tumor volume of Cx43G21R group increased,and after administration of dioscin,the tumor volume decreased significantly in a concentrationdependent manner.Immunohistochemistry showed that the expression of Cx43 protein in Cx43G21R group was higher than that in Vector group,and the expression of dioscin was further increased after administration of dioscin.Compared with Vector group,the expression of Ecadherin protein in Cx43G21R group decreased,while that in dioscin group increased,while the expression level of N-cadherin protein increased significantly in Cx43G21R group,and decreased in a concentration-dependent manner after intervention with dioscin.The results of Western Blot showed that compared with Ctrl group,the expression of p-PI3K,p-Akt and pmTOR protein in Cx43G21R group increased and decreased significantly after treatment,but there was no significant difference in the expression of PI3K,Akt and mTOR protein.The expression of E-cadherin and N-cadherin protein was the same as that of immunohistochemistry.Conclusion:Dioscin inhibits mTOR/PI3K/Akt signal pathway by upregulating the gap junction communication function mediated by Cx43,so as to achieve the effect of anti-gastric cancer epithelial-mesenchymal transformation.