| Peritoneal metastasis of gastric cancer seriously affects the prognosis of patients with advanced gastric cancer.Although the "seed-soil" theory is the generally accepted theory of peritoneal metastasis in gastric cancer,the internal mechanism of cancer metastasis is not fully uncovered.Protein is the implementation of all life activities.To find the key proteins in peritoneal metastasis of gastric cancer,we searched the differentially expressed proteins among normal gastric epitheliums,primary gastric cancer tissues and peritoneal metastases by two-dimensional gel electrophoresis and mass spectrometry,and found myosin IIA(MYH9)as the highest disregulated protein which was further confirmed by western blot and TCGA data analysis.In addition,we also found that the expression of MYH9 was negatively correlated with the prognosis of gastric cancer.Secondly,we found the mRNA and protein levels of β-catenin were down-regulated as MYH9 was knocked down by lentivirus shRNA interference and TALEN in gastric cancer cell lines.Further study showed MYH9 largely located in the nucleus by using confocal microscopy,and nuclear MYH9 protein was found to target to the AGCTCC sequence,which was located in the promoter of β-catenin,by its LEKAKQTLENERGELANEVKVL sequence and promoted the transcription of β-catenin by software analysis of nuclear localization signal(NLS),CoIP and Luciferase Reporter Gene Assay.Moreover,we found that MYH9/β-catenin axis could increase the anoikis resistance of gastric cancer cells by assays of cell apoptosis,adhesion and anoikis.Lastly,we found that phosphorylation of MYH9 protein at 1943S contributed to the regulation of CTNNB1 transcription.Staurosporine,which could inhibit the 1943S phosphorylation of MYH9 protein and subsequent CTNNB1 transcription,decreased the metastasis of gastric cancer in orthotropic murine models.In conclusion,this study found that MYH9,known as cytoplasmic myosin Ⅱa,also located in the nucleus of gastric cancer cells and regulated the transcription of β-catenin,increased the anoikis resistance of gastric cancer cells and promoted peritoneal metastasis of gastric cancer.The subject consisted of four parts.The specific contents are as follows:Chapter 1:MYH9 located in the nucleus and increased the transcription level of CTNNB1 in gastric cancer cell linesObjective:To explore the mechanism of key proteins in peritoneal metastasis and its molecular mechanism by proteomics.Methods:Two-dimensional gel electrophoresis-mass spectrometry,qPCR and western blot,immunohistochemistry(tissue microarray),TCGA data analysis,nuclear protein CoIP,cell immunofluorescence confocal detection;MYH9 knockdown and overexpression stable strain construction(TALEN,lentivirus shRNA interference and lentivirus MYH9 overexpression)Results:MYH9 was upregulated in peritoneal metastatic tissues compared with normal gastric epithelium and primary cancer tissues.Knocking down the expression of MYH9 down-regulated the expression of β-catenin in gastric cancer cell lines.The nuclear MYH9,β-actin,myosin light chain MYL9 and transcription factor TFIIB co-located in the nucleus.Chapter 2:Nuclear MYH9 promoted the transcription of CTNNB1 gene.Objective:To explore the molecular mechanism of MYH9 in the transcription of CTNNB1.Methods:MYH9 nuclear localization signal site prediction and cell plasmid transfection;truncated plasmid construction and reporter gene assay;MYH9 computer 3D protein model construction and research on its interaction with DNA modelResults:The MYH9 amino acid sequence KRKK at position 1246-1249 was the key signal for MYH9 nuclear localization.MYH9 promoted β-catenin transcription by recognizing and binding to the β-catenin promoter region AGCTCC sequence.MYH9 protein bound to β-catenin promoter AGCTCC by LEKAKQTLENERGELANEVKVL Sequence.Phosphokinase inhibitor Staurosporine inhibited MYH9-mediated CTNNB1 transcription by affecting phosphorylation of MYH9 protein at position 1943S.Staurosporine could effectively inhibit the growth and peritoneal metastasis of gastric cancer in orthotropic murine models.Chapter 3:MYH9/β-catenin axis promotes the anoikis resistance and peritoneal metastasis of gastric cancerObjective:To explore the mechanism of MYH9/β-catenin signal axis in promoting peritoneal metastasis of gastric cancerMethods:Cell transfection,apoptosis test,adhesion test and tumor anoikis resistance detection;orthotropic murine models construction and Staurosporine intraperitoneal injectionResults:MYH9-mediated β-catenin expression reduced early cell apoptosis,increased cell adhesion and the anti-tumor ability of gastric cancer cells(anoikis resistance),and lastly promoted the peritoneal metastasis of gastric cancer.Staurosporine effectively inhibited the metastasis of gastric cancer orthotropic murine models.By doing this study,we could get the following conclusions.Firstly,peritoneal metastasis was related to the upregulation of MYH9 expression.Secondly,MYH9 located in the nucleus,promoted the CTNNB1 gene transcription and gastric cancer cells’ anoikis resistance.The innovation of this study is found nuclear MYH9 increased the transcription of CTNNB1 and inhibited anoikis of gastric cancer cells. |
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