Research On The Effect And Mechanism That Ziyin Huatan Recipe Inhibits Metastasis Of Gastric Cancer

Objective:Gastric cancer（GC）is the major leading cause of cancer-related mortality in China.Surgery is currently the curative treatment for GC;chemotherapy,radiotherapy and even gene therapy are considered the main adjuvant-therapy methods.However,accounting for the late detection,drug resistance and toxic side effects,new therapies should be developed to improve the prognosis of GC.Ziyin Huatan Recipe（ZYHT）developed for the advanced gastric cancer,was based on hypothesis of "tumor-phlegm microenvironment" and had shown its promising value in clinic.However,the potential role of ZYHT in the treatment of GC and the precise mechanisms have not yet been clearly addressed.Therefore,this study aimed to predict targets and molecular mechanisms of ZYHT by network pharmacology analysis and to evaluate the role of ZYHT on gastric cancer by in vitro and in vivo studies.Methods:1.Screen the main components of ZYHT from network database and literature.Predict its anti-tumor targets.Perform gene ontology（GO）biological process and KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway analysis of anti-tumor targets of ZYHT using DAVID database.2.The influence of ZYHT with different concentrations on the proliferation of GC cell lines such as SGC-7901 and MGC-803 was measured by Cell Counting Kit-8（CCK-8）.3.Appropriate concentrations of ZYHT was selected from the above experimental results.The influence of ZYHT on the migration and invasion of SGC-7901 and MGC-803 cells was detected through wound healing assay and Transwell chamber migration and invasion assay.4.In SGC-7901 and MGC-803 cells,the effect of ZYHT on expression of MMP2,MMP9,Snail,Slug,Vimentin,E-Cadherin,N-Cadherin and RUNX3,which were predicted through network pharmacology analysis,were explored by Western blot（WB）and Quantitative Real-Time PCR（qPCR）.5.To explore the specific molecular mechanisms of ZYHT on the migration and invasion,RUNX3 gene was knocked out by CRISPR/Cas9 and the lentiviral vectors were transfected into SGC-7901 cells.Wound healing and Transwell chamber assay were conducted.WB was used to detect the protein change of E-Cadherin,N-Cadherin,Vimentin,MMP2 and MMP9.6.To explore the ability of ZYHT in suppressing GC cells metastasizing to lung,we established a lung metastasis model of gastric cancer in nude mice.WB and immunohistochemistry（IHC）were used to explore the impact of ZYHT on the expression of metastasis-related proteins whether RUNX3 gene was knocked out or not.7.The influence of ZYHT on angiogenesis was measured with tube formation assay of HUVECs using co-culture supernatant of ZYHT and GC cells including SGC-7901 and MGC-803 cells.To explore the effect of ZYHT on angiogenesis in vivo,a subcutaneous xenograft model of GC in nude mice was constructed and IHC was used to detect the microvessel density（MVD）in the tumor tissue.8.The effects of ZYHT on expression of VEGFA and VEGFC in GC cells,cell supernatants,tumor-bearing nude mice blood and tumor tissues after treated with ZYHT were detected by Enzyme-Linked ImmunoSorbent Assay（ELISA）,q-PCR and WB.Results:1.The results of network pharmacology analysis showed that ZYHT might have influence on several GO biological processes,such as extracellular matrix disassembly,positive regulation of epithelial to mesenchymal transition,positive regulation of cell migration,positive regulation of focal adhesion assembly,negative regulation of metallopeptidase activity and response to wounding and angiogenesis.2.As CCK-8 assay showed,ZYHT may evidently inhibit the proliferation of SGC-7901 and MGC-803 cells in a concentration-dependent manner.However,when the treatment concentration was less than 100μg/mL,ZYHT had little effect on the proliferation of SGC-7901 and MGC-803 cells.Therefore,in subsequent experiments,25μg/mL,50μg/mL and 100μg/mL of ZYHT were selected as low,middle,and high concentration for further studies in vitro.3.Wound healing and Transwell chamber assay show that ZYHT inhibits the migration and invasion of SGC-7901 and MGC-803 cells in a dose-dependent manner when ZYHT concentration was less than 100μg/mL.4.The results of WB and q-PCR showed that the expression of MMP2,MMP9,N-Cadherin and Vimentin were significantly decreased by treating with ZYHT in a concentration-dependent manner,whereas the tendency of RUNX3 and E-Cadherin expression were inverse.However,the expression of Snail and Slug were remained essentially unchanged.5.We successfully constructed SGC-7901-RUNX3^-/- cells.Wound healing and Transwell chamber assay showed that ZYHT may inhibit the migration and invasion of GC cells by up-regulating the expression of RUNX3.Compared with normal SGC-7901 cells,the protein expression of N-Cadherin,Vimentin,MMP2 and MMP9 in SGC-7901-RUNX3^-/- cells were up-regulated by ZYHT,whereas the tendency of E-Cadherin expression was inverse.6.The lung-metastasis model of gastric cancer in nude mice was successfully constructed with SGC-7901 and SGC-7901-RUNX3^-/- cells.Results showed that ZYHT might inhibit the lung metastasis ability of SGC-7901 cells compared with the saline control group.ZYHT can inhibit the lung metastasis ability of normal SGC-7901 cells more efficiently than SGC-7901-RUNX3^-/- cells.In addition,compared with normal group,the survival time of ZYHT-treated nude mice was significantly prolonged.The results of WB and IHC showed that the effects od ZYHT on lung metastasis tissue in vivo were in accordance with that in vitro.7.The co-culture supernatant of ZYHT and GC cells could significantly inhibit the tube formation ability of HUVECs.In vivo,ZYHT may reduce the microvessel density in the tumor tissue of subcutaneous xenografts in nude mice.8.The results of ELISA,q-PCR and WB showed that ZYHT may reduce the expression of VEGFA and VEGFC in GC cells,cell supernatants,tumor-bearing nude mice blood and tumor tissues.Conclusion:1.ZYHT can inhibit the invasion and migration of GC in vitro and in vivo,and its molecular mechanism may relate to the up-regulation of RUNX3 expression.2.ZYHT has an anti-angiogenic effect on GC in vivo and in vitro,and its molecular mechanism may relate to the down regulation of VEGFA and VEGFC expression.