MicroRNA-647 Targets SRF-MYH9 Axis To Suppress Invasion And Metastasis Of Gastric Cancer

MicroRNAs(miRNAs)play important roles in regulating tumour development and progression.Here we show that miR-647 is repressed in gastric cancer(GC),and associated with GC metastasis.Moreover,we identify that miR-647 can suppress GC cell migration and invasion in vitro.Mechanistically,we confirm miR-647 directly binds to the 3' untranslated regions of SRF mRNA,and SRF binds to the CArG box located at the MYH9 promoter.CCG-1423,an inhibitor of RhoA/SRF-mediated gene transcription,inhibits the expression of MYH9,especially in SRF downregulated cells.Overexpression of miR-647 inhibits MGC 80-3 cells' metastasis in orthotropic GC models,but increasing SRF expression in these cells reverses this change.Importantly,we found the synergistic inhibition effect of CCG-1423 and agomir-647,an engineered miRNAmimic,on cancer metastasis in orthotropic GC models.Our study demonstrates that miR-647 functions as a tumor metastasis suppressor in GC by targeting SRF/MYH9 axis.This topic is composed of three parts.The specific content is as follows:Chapter 1:Down-regulation of MicroR-647 expression in gastric cancer inhibits gastric cancer cell metastasisObjective:To verify the correlation between microR-647 expression and clinicopathological parameters in gastric cancer,and to verify the effect of microR-647 expression on the invasion and metastasis of gastric cancer cells.Methods:qPCR,lentiviral transfection-mediated microR-647 overexpression,transient transfection mediated-microR-647 overexpression and knockdown,transwell chamber and scratch cytology experiment.Results:The expression of micron-647 in gastric cancer tissues was significantly down-regulated.The expression of microR-647 was negatively correlated with TNM stage,T stage and N stage.The expression of microR-647 in gastric cancer cells inhibited the invasion and metastasis of gastric cancer cells.Chapter 2:MicroR-647 regulates SRF/MYH9 expression to inhibit Gastric Cancer Metastasis in gastric cancerObjective:To explore the molecular mechanism by which microR-647 inhibits gastric cancer cell metastasis.Methods:Cell transfection,WB,qPCR,IHC,in situ hybridization(ISH),CoIP,ChIP,construction of truncated plasmids and detection of luciferase reporter genes were performed.RESULTS:Rho pathway-associated min-647 inhibited SRF and MYH9 expression in gastric cancer cells.MiR-647 directly inhibited SRF expression,indirectly inhibited MYH9 mRNA,and attenuated gastric cancer cell metastasis and invasion.MiR-647,SRF and MYH9 expression levels were associated with poor prognosis of gastric cancer patients.CCG-1423 is effective in inhibiting gastric cancer cell metastasis.Activation of Rho/SRF/MYH9 signaling pathway axis promotes metastasis of gastric cancer and suggests poor prognosis of gastric cancer patients.Chapter 3:MicroR-647 associated tumor therapy for orthotopic implanted tumor in nude mice.OBJECTIVE:To study the effect of the MicroR-647/SRF/MYH9 axis on the metastasis of orthotopic implanted tumor in nude mice and the treatment effect of CCG-1423 and agomir-647 on tumors in vivo.Methods:Stomach cancer implanted in nude mice.CCG-1423 and agomir-647 drug intervention,immunohistochemistry and HE staining.RESULTS:MicroR-647 inhibited orthotopic implanted tumor metastasis of gastric cancer in nude mice by SRF/MYH9 signalling.Combined using CCG-1423 and agomiR-647 can effectively inhibit the metastasis of gastric cancer in orthotopic implanted tumor model in nude mice.From the above studies,we have concluded that miR-647 is downregulated in gastric cancer and is associated with metastasis.miR-647 targets the 3' untranslated region of SRF mRNA and inhibits SRF expression.SRF binds to the CArG box of the MYH9 promoter to promote its expression,and promote invasion and metastasis of gastric cancer.CCG-1423 inhibits SRF-mediated MYH9 expression.Combined use of CCG-1423 and agomir-647 can effectively inhibit gastric cancer metastasis in orthotopic implanted tumor models in nude mice.The innovation of this study:we confirmed the correlation between miR-647/SRF/MYH9 axis and gastric cancer metastasis.It was found that the combination use of CCG-1423 and agomir-647 inhibited the metastasis of gastric cancer in nude mice orthotopic implanted tumor models,which provided new ideas for clinical practice.