Molecular Mechanism Of TRIM44 Mediating Gastric Cancer Stem Cell Self-renewal And Tumorigenesis

Background: Gastric cancer is a kind of malignant tumor with high morbidity and mortality.Although the popularization of early screening and advances in diagnosis and treatment strategies have improved the prognosis of gastric cancer patients to a certain extent,the long-term prognosis of gastric cancer patients is still low.The main factors causing the poor prognosis of gastric cancer patients are recurrence,metastasis and chemo-resistance.Gastric cancer stem cells refer to a small number of cell subpopulations with self-renewal ability and differentiation potential which exist in gastric cancer.It is generally believed that gastric cancer stem cells are one of the fundamental reasons for the recurrence,metastasis and drug resistance of postoperative patients.Existing studies have highlighted the role of protein ubiquitination and deubiquitination processes in regulating cancer stem cell self-renewal and differentiation.Members of the tripartite motif protein(TRIM)family are key players in the process of protein ubiquitination and deubiquitination and have been reported to contribute to the maintenance of the stemness characteristics of cancer stem cells.However,the biological functions of TRIM44 which was one of the TRIM family have not yet been reported in gastric cancer and gastric cancer stem cells.This study aims to explore the role of TRIM44 in gastric cancer and gastric cancer stem cells using bioinformatics,clinical tissue samples and molecular biology experiments,so as to expand the understanding of gastric cancer occurrence,development and treatment strategies.Methods: Bioinformatics and biological experiments(RT-q PCR,immunohistochemistry and western blotting)were used to detect the expression level of TRIM44 in gastric cancer,and combined with clinical follow-up data to analyze the prognostic value of TRIM44 in gastric cancer patients.By constructing stably downregulation or overexpression TRIM44 cell lines,the effects of TRIM44 on the proliferation and metastasis of gastric cancer cells were explored by plate cloning,wound healing,Transwell,nude mouse tumorigenesis and lung metastasis model experiments.The expression level of TRIM44 was detected after inducing the formation of gastric cancer stem cell microspheres by conditioned culture.Subsequently,the role of TRIM44 in gastric cancer stem cell selfrenewal and tumorigenesis was analyzed by in vitro spheroidization,flow cytometry experiments and in vivo gradient dilution tumorigenesis experiments.Experiments such as co-immunoprecipitation,mass spectrometry,immunofluorescence co-localization,western blotting and immunohistochemistry were used to further explore the potential molecular regulation mechanism of TRIM44 in gastric cancer.In addition,we also explored the role of TRIM44 in regulating the chemosensitivity of gastric cancer through in vivo and in vitro chemotherapy models.Results: The expression level of TRIM44 in gastric cancer tissues was significantly higher than that in adjacent normal tissues,and the long-term prognosis of patients with high TRIM44 expression was significantly worse than that of patients with low TRIM44 expression.TRIM44 can promote the proliferation and migration of gastric cancer cells in vitro and it can also promote subcutaneous tumor formation and lung metastasis in vivo.In addition,we also found that the expression level of TRIM44 in gastric cancer stem cells was significantly increased,and the high expression of TRIM44 could promote the self-renewal of gastric cancer stem cells and enhance gastric cancer tumorigenesis.Mechanistically,TRIM44 binds to 14-3-3? through the B-box domain and deubiquitinates14-3-3? through the zinc finger domain to prevent 14-3-3? from protein degradation via the ubiquitin-proteasome pathway.14-3-3? accumulation leads to upregulation of ?-catenin,which in turn enhances the transcription and activation of cancer stemnessrelated factors such as Lgr5,c-Myc,SOX2 and SOX9.Finally,we also demonstrated that inhibition of TRIM44 enhanced the chemosensitivity of gastric cancer cells.Conclusion: We demonstrated that the TRIM44/14-3-3?/?-catenin signaling axis can enhance gastric cancer stem cell self-renewal and tumorigenesis.TRIM44 is a potential target for developing molecularly targeted drugs.