TRIM2 Suppresses Proliferation And Migration Of Clear Cell Renal Cell Carcinoma Via Downregulation Of β-catenin

Background and Objective:Renal cell carcinoma(RCC)accounts for about 2% of all malignant tumors in adult,among which,clear cell renal cell carcinoma(ccRCC)accounts for 80% to 85% of allrenal cell carcinoma,and its incidence has been increased in recent decades.Statisticsshow that,about 209,000 new cases and 102,000 deaths from RCC annually.At the initialdiagnosis,20-30% of patients have metastatic ccRCC and the recurrence rate ofpost-surgery is about 30%,and metastasis remains the main reason for RCC-associatedmortality.Besides,ccRCC is insensitive to chemo/radiotherapies and targeted therapeuticoptions.Therefore,it’s urgent to explore new genes that provide new clues and strategiesfor revealing the molecular mechanism,diagnosis and treatment of ccRCC.In the study,we tested the expression of TRIM2 in renal tumor tissues and normaltissues of ccRCC,compared TRIM2 expression levels at different stages and grade andmade the Kaplan-Meier survival analysis of ccRCC.Besides,we evaluated theproliferation and migration of TRIM2 in ccRCC.To further investigate its underlyingmechanisms,the expression of important markers of migration in ccRCC cells was tested.The purpose of this study is to provide a new molecular target for the diagnosis andtreatment of ccRCC.Methods:1.Immunohistochemical staining of tissue microarrays was to test the expression of TRIM2 in renal tumor tissues and normal tissues from 50 patients of ccRCC.2.We investigated the mRNA expression of TRIM2 in ccRCC tissues and normal subjects,compared TRIM2 expression levels at different stages and grade and made the Kaplan-Meier survival analysis of ccRCC patients from UALCAN database(http://ualcan.path.uab.edu/index.html).3.The plasmid which carries TRIM2 cDNA was transfected to two ccRCC cell lines(786-O and 769-P),and cck8 assay and plate clone formation assay were performed to test cells proliferation.4.786-O and 769-P cells which were transfected TRIM2 cDNA and control cDNA were to evaluate the ablity of tumor cells migration by wound healing assay and tranwell assay.5.To further investigate whether TRIM2 affected the migration of cancer cells and its underlying mechanisms,the expression of important markers of migration in ccRCC cells was detected by western blot and immunoprecipitation.Results:1.There was 78% lower expression of TRIM2 in renal tumor tissues than in normal subjects from 50 patients of ccRCC tissue microarray.2.In the UALCAN,the expression of TRIM2 mRNA in normal subjects was higher than in primary tumor(P<0.001),and was higher in normal subjects than other 4 stages and grade(P<0.001),indicating that TRIM2 may be a tumor suppressor in ccRCC.The Kaplan-Meier survival analysis showed that the ccRCC patient group with high level of TRIM2 had a better survival possibility than the group with low/medium level of TRIM2(P<0.001).3.The level of TRIM2 was significantly upregulated in 786-O and 769-P cells after transfected with the TRIM2 over-expression plasmid with HA-tag.Western blot and q-PCR demonstrated that TRIM2 over-expression plasmid with HA-tag was transfected to two ccRCC cell lines successfully.CCK8 assay indicated that over-expression of TRIM2 led to a significant inhibition of the tumor cell growth(P<0.001),and the ability of colony formation also decreased in 786-O(P<0.01)and 769-P(P<0.001)cells transfected with TRIM2.Together,TRIM2 can attenuate ccRCC proliferation in vitro.4.The motility of 786-O and 769-P cells with TRIM2 over-expression was detected by wound healing and transwell assays.Wound healing assay showed that the over-expression of TRIM2 significantly inhibited cells migration in two ccRCC cells(P<0.001),and transwell assays also illustrated that the number of cells was decreased after TRIM2 transfection in both cells(P<0.001).Taken together,TRIM2 inhibited the ability of cell motility in ccRCC cells in vitro.5.we found that TRIM2 could decreaseβ-catenin.786-O and 769-P cells transfected with increasing amount of TRIM2 cDNA could decrease the expression ofβ-catenin gradually.In the transfection experiment in 293-T cells,TRIM2 could not only degradeβ-catenin,but also bind to it.Conclusions:1.The lower expression of TRIM2 in renal tumor tissues than in normal subjects.2.the expression of TRIM2 mRNA in normal subjects was higher than in primary tumor,and was higher in normal subjects than other 4 stages and grade.The ccRCC patient group with high level of TRIM2 had a better survival possibility than the group with low/medium level of one.3.TRIM2 suppressed ccRCC cells proliferation and migration via degradation of β-catenin.