Studies On The Mechanism Of MiR-10b-5p And Cullin3-SPOP Regulating TIAM1 To Inhibit The Proliferation,Migration And Invasion Of Gastric Cancer

Gastric cancer(GC)is one of the most common malignant tumors of the digestive system.According to the 2020 global cancer statistics,there were more than1 million new cases and an estimated 769,000 deaths.Due to the lack of typical clinical symptoms and biomarkers for detection and treatment of early gastric cancer,some patients are diagnosed at an advanced stage.Therefore,exploring the molecular mechanism of the occurrence and metastasis of gastric cancer,and finding the molecular biomarkers that can effectively predict gastric cancer are still crucial for improving the diagnosis rate and prognosis of gastric cancer patients.It has been confirmed that T-lymhom invasion and metastasis gene 1(TIAM1)is closely related to the proliferation,migration and invasion of gastric cancer.However,the current research on the regulation of TIAM1 activity is insufficient.Previous experiments demonstrated that miR-10b-5p inhibited the gastric cancer progression by down regulating TIAM1 expression in vitro.However,it is not clear whether it also inhibits the proliferation and metastasis of gastric cancer in vivo.Additionally,previous studies have shown that TIAM1 protein is regulated by the ubiquitin proteasome system.E3 ubiquitin ligase substrate adaptor protein Speckle-type POZ domain protein(SPOP)can bind and ubiquitinate to degrade substrate proteins through the SPOP-binding consensus(SBC)region.Mutations in the SPOP gene or abnormal protein expression is associated with the occurrence and progression of various cancers.The amino acid sequence of TIAM1 protein contains an SBC region,so we hypothesized that SPOP can regulate the activity of TIAM1 protein by ubiquitination,thereby affecting the biological behavior of gastric cancer.In order to determine that miR-10b-5p and SPOP affect the proliferation and metastasis of gastric cancer by regulating the expression and activity of TIAM1,we first used dual luciferase assay,clone formation assay,CCK-8 proliferation assay and scratch healing assay to verify miR-10b-5p inhibits the proliferation and migration of gastric cancer cells by targeting down-regulation of TIAM1 expression in cultured cells.In addition,by constructing a nude mouse model of gastric cancer xenografts,it was confirmed that miR-10b-5p could significantly inhibit the proliferation and metastasis of gastric cancer xenografts in vivo.Then,we demonstrated that Cullin3-SPOP ubiquitination regulates TIAM1 protein by co-immunoprecipitation assay,Ni-NTA ubiquitin assay,protein degradation assayand protein half-life assay.In human gastric tissue samples,the mRNA and protein expressions of TIAM1 and SPOP in cancer and adjacent normal tissues were detected by RT-q PCR and Immunohistochemical staining,and the correlation between the expression of the two and their correlation with TNM staging,lymph node metastasis in gastric cancer was analyzed.The results showed that In gastric cancer tissue,the mRNA and protein expression levels of SPOP in gastric cancer tissue were lower than those in normal tissue 5 cm adjacent to the tumor,and were negatively correlated with the expression of TIAM1.In addition,gastric cancer cells stably expressing wild-type and mutant SPOP were constructed by lentiviral packaging.Clone formation experiments,CCK-8proliferation experiments,scratch healing experiments,and Transwell invasion experiments were performed.The results showed that SPOP mutation resulted in stronger proliferation,migration and invasion ability of gastric cancer cells compared with wild-type SPOP.Furthermore,gastric cancer cells had a reduced number of colonies,a reduced rate of proliferation,a reduced rate of wound healing,and a significant reduction in the number of cells traversing the Transwell Matrigel membrane after stable knockdown of TIAM1 in SPOP mutant cell lines compared with controls(all differences above has statistical significane).This indicated that knockdown of TIAM1 inhibited the proliferation,migration and invasion of SPOP mutant gastric cancer cell lines.Taken together,the results of this study indicated that miR-10b-5p inhibits the proliferation and migration of gastric cancer cells by down-regulation of the expression of TIAM1.In addition,Cullin3-SPOP modulates the activity of TIAM1 by ubiquitination,thereby inhibiting the proliferation and migration of gastric cancer cells.The innovation of this study is verified that miR-10b-5p inhibits the progression of gastric cancer by down-regulation TIAM1 in vivo,and proved that TIAM1 protein is a new substrate of SPOP.The significance of this study is to elucidate the new mechanism of TIAM1 regulation affecting the proliferation,migration and invasion of gastric cancer,and to provide new biomarkers and targets for the clinical diagnosis and treatment of gastric cancer.