The Role And Molecular Mechanism Of USH1G On Migration And Invasion In Gastric Cancer

Objective 1.To investigate the expression of USH1 G in gastric cancer and and its influence on prognosis of gastric cancer;2.To reveal the effects of USH1 G on migration and invasion in gastric cancer;3.To investigate the mechanism of USH1 G in gastric cancer.Methods 1.The protein levels of USH1 G in 48 gastric cancer and corresponding adjacent tissues were detected by Western Blot.The bioinformatics technique was used to analyze the relation between USH1 G m RNA and prognosis in gastric cancer cases via TCGC database.2.USH1 G over-expression stable cell line and knock-down stable cell line were established by lentivirus transfection.3.To detect the effects of USH1 G on the invasion,migration and adhesion of gastric cancer cells,the wound healing,transwell and cell adhesion assay were performed.4.Immunofluorescence were performed to observe the effect of USH1 G on the filopodia of gastric cancer cells.5.The effects of USH1 G on EMT markers and Rho-ROCK pathway-related proteins were determined by quantitative real time polymerase chain reaction(q PCR),Western Blot and bioinformatics analysis.6.USH1 G and MYH9 were identified as interacting proteins by immunoprecipitation,protein mass spectrography and Western Blot,and the interaction domain was determined.7.To determine the effect of USH1 G on MYH9 protein level,Western Blot was performed,and the expression of MYH9 in 48 cases of gastric cancer and corresponding adjacent tissues were detected as well.8.Determining the down-regulation of MYH9 protein level by USH1 G through the ubiquitin-proteasome pathway by immunoprecipitation and Western Blot.9.Western Blot and immunofluorescence were performed to determine that USH1 G repressed MYH9 protein levels via the autophagy-lysosomal pathway.Results 1.The protein level of USH1 G in gastric cancer tissues was significantly lower than that in benign tissues,which was related to the poor prognosis.2.USH1 G inhibited the migration,invasion and adhesion of gastric cancer cells.3.USH1 G reduced the number of filamentous pseudopods in gastric cancer cells.4.MYH9 and USH1 G were interacting proteins,and the SAM domain of USH1 G was a necessary domain.5.USH1 G down-regulated the MYH9 protein in gastric cancer cells,and the level of MYH9 protein in gastric cancer tissues was significantly higher than that in adjacent tissues.6.USH1 G suppressed MYH9 protein levels through ubiquitin-proteasome pathway.7.USH1 G down-regulateed MYH9 protein levels via the autophagy-lysosomal pathway as well.Conclusion The protein level of USH1 G in gastric cancer tissues was significantly lower than that of benign tissues,which was associated with poor prognosis.USH1 G inhibited the migration,invasion and adhesion of gastric cancer cells and reduced the number of filamentous pseudopods in gastric cancer cells.USH1 G downregulated the level of the interacting protein MYH9 protein via the ubiquitin-proteasome pathway and the autophagy-lysosomal pathway.