Mechanism Study Of Deubiquitinase TRIM44 Regulating The Progression Of Gastric Cancer

Background:Gastric cancer(GC)is one of the most common malignant tumors worldwide.Due to the lack of effective treatment for GC,the patients of gastric cancer usually with a poor prognosis.In the recent years study,Deubiquitinating enzyme plays an important role in tumor progression.Ubiquitination modification is a reversible process,deubiquitinase can hydrolyze the connection between ubiquitin molecules and substrate proteins,and it’s abnormal expression plays a driving role in the development of tumors.Tripartite Motif Containing 44(TRIM44)contains zinc finger structure,which plays an important role in protein stability.Current research shows that TRIM44 is up-regulated in many tumors,which affects tumor progression and is associated with poor prognosis.However,the regulatory mechanism of TRIM44 in gastric cancer and its impact on the prognosis of gastric cancer are still unexplained.Tumor progression is a complex process,and the abnormal biological behavior of tumor cells involves a variety of signal pathways and gene mutation.Therefore,investigating and understanding the regulatory mechanism of deubiquitinase for GC will help to find new tumor markers,which is of great significance for the diagnosis of gastric cancer and the treatment for patients.Methods:In the early stage,we analyzed the expression of TRIM44 in gastric cancer and adjacent to the cancer with TCGA database,draw survival curve and carried out single factor and multi factor survival regression analysis.Real time quantitative PCR was used to verify TRIM44 relative expression in GC sample;The expression of TRIM44 in GC tissue was verified by immunohistochemistry.The effect of TRIM44 expression on the migration and proliferation of gastric cancer cells was explored by Transwell migration experiment and CCK8 cell proliferation experiment.The substrate protein of TRIM44 was screened by CO-IP experiment and mass spectrometry analysis;The interaction between TRIM44 and its substrate protein was verified by CO-IP experiment,immunofluorescence co-localization,western blot.The effect of TRIM44 expression on mitochondrial morphology was observed by confocal microscopy,and the localization of the substrate protein regulated by TRIM44 and mitochondria was also observed.The metabolic transformation of tumor cells was verified by lactate production experiment,ATP generation experiment and OCR oxygen consumption rate experiment.Western blot experiment verified the regulation of TRIM44 on downstream target genes and the regulation of signal pathways that promote the migration and proliferation of gastric cancer cells after metabolic transformation.By using TCGA database analysis,TRIM44 had promoter methylation modification and predicted methylation sites.The methylation inhibitor 5-Azacytidine and western blot experiments were used to identify the methyltransferase that mediates the methylation modification of TRIM44 promoter region.Results:(1)The expression of TRIM44 in gastric cancer and its clinicopathological significanceTRIM44 is up-regulated in gastric cancer.Patients with high TRIM44 expression have poor prognosis.(2)TRIM44 promotes the migration and proliferation of gastric cancer cellsIn vitro,cell function experiment showed that up-regulated expression of TRIM44 enhanced the migration and proliferation of gastric cancer cells.(3)TRIM44 stabilizes TRAP1 protein expression by regulating its ubiquitinationTRIM44 and TRAP1 are co-located in the cytoplasm and interact with each other.TRIM44 plays the role of deubiquitinase to remove the ubiquitination modification of TRAP1,thus stabilizing its protein level.(4)TRIM44 regulates mitochondrial morphology and promotes migration induced metabolic reprogramming in gastric cancer cellsTRIM44 regulates DRP1 leading to mitochondrial division,and stabilize TRAP1 expression making aerobic glycolysis the main metabolic form of tumor cells by changing the function of mitochondria;Mitochondrial division enhances the migration ability of gastric cancer cells.(5)DNA methylation involved in regulation of TRIM44 expressionTRIM44 has promoter methylation modification.Methyltransferase DNMT1 participates in the regulation of TRIM44 methylation modification,controlling its transcription level.Conclusions:1.TRIM44 is highly expressed in gastric cancer and is associated with poor prognosis.2.TRIM44 expression enhances the proliferation ability of gastric cancer cells.3.TRIM44 regulates the ubiquitination of TRAP1 and stabilizes its protein expression.4.TRIM44 induces mitochondrial division also enhances the migration ability of gastric cancer cells,and makes aerobic glycolysis the main intracellular metabolic form.5.Methyltransferase DNMT1 is involved in the regulation of TRIM44 transcription by DNA methylation.