| Objective:Ovarian cancer(OC)is one of the most common and dangerous gynecological malignant tumors.Due to its late onset of clinical symptoms,high rate of metastasis and recurrence,it is difficult to detect early,and the 5-year survival rate is less than 50%.Serous ovarian cancer is the most common pathological type of epithelial ovarian cancer and has the highest degree of malignancy.Serous ovarian cancer has the worst prognosis and the lowest 5-year survival rate,so it is particularly important to study the occurrence and development mechanism of serous ovarian cancer.The discovery of a new mechanism of action is helpful for the early diagnosis and treatment of serous ovarian cancer,and provides a reference for the improvement of clinical treatment strategies,so as to improve the prognosis of serous ovarian cancer.Ubiquitination is one of the important ways of protein homeostasis regulation in cells,and it is involved in many biological behaviors such as transcriptional regulation,DNA repair and cell proliferation.In addition,ubiquitination has been found to be involved in the occurrence and development of various tumors,including ovarian cancer.Therefore,the study of the role of ubiquitination in the development of serous ovarian cancer is helpful to explore potential therapeutic targets.TRIM22,a member of the tripartite motif-containing proteins(TRIM)family,has E3 ubiquitin ligase activity due to its RING domain and is responsible for the specific recognition of substrate proteins during ubiquitination.Early studies focused on the immune system,and in recent years TRIM22 has been found to play different biological effects in different tumors,such as tumor suppressive effects in endometrial cancer and osteosarcoma,and tumor promoting effects in glioblastoma and non-small cell lung cancer.However,its expression and function in serous ovarian cancer remain unclear.transcription factor 4(TCF4)is one of the important transcription factors of the T cell factor/lymphatic enhancer(TCF/LEF)family,which can bind related proteins in the nucleus,activate downstream signaling pathways,and then regulate target genes to affect cell proliferation and differentiation and other biological functions.Studies have shown that TCF4 plays a carcinogenic role in epithelial ovarian cancer and is an independent biomarker to judge the prognosis of epithelial ovarian cancer.However,the regulatory effects of TRIM22 on TCF4 have not been reported in the specific histogenetic context of serous ovarian cancer.Therefore,this study aims to explore the molecular mechanism related to the occurrence and development of serous ovarian cancer caused by E3 ubiquitin ligase TRIM22,so as to lay the molecular basis for the occurrence and development of serous ovarian cancer.At the same time,the interaction mechanism between TRIM22 and target protein TCF4 was explored through relevant studies to provide a scientific and powerful reference for effective clinical treatment of serous ovarian cancer.Methods :Part Ⅰ: To predict TRIM22 mRNA expression in serous ovarian cancer and normal ovarian tissue using online data at http://gepia.cancer-pku.cn/index.html.The expression of TRIM22 mRNA in serous ovarian cancer and normal ovarian tissue was detected by RT-PCR.Western Blot and immunohistochemical methods were used to detect the expression of TRIM22 protein in serous ovarian cancer tissue and normal ovarian tissue,and to analyze the relationship between TRIM22 protein and the clinicopathologic characteristics and survival prognosis of patients with serous ovarian cancer.The expression of TRIM22 mRNA and protein in 6 ovarian cancer cell lines(OVCAR3,SKOV3,SW626,HO-8910,A2780 and Co C1)was detected by RT-PCR and Western Blot,respectively.The effects of TRIM22 on the proliferation of OVCAR3,SKOV3 and A2780 serous ovarian cancer cells were detected by CCK-8 and clonal formation assay.Meanwhile,the effects of underexpression and overexpression of TRIM22 protein on proliferation-related proteins PCNA,Cyclin D1 and Cyclin E in SKOV3 and A2780 serous ovarian cancer cells were detected by Western Blot.Transwell and scratch assay were used to detect the effect of TRIM22 on the migration and invasion ability of OVCAR3,SKOV3 and A2780 serous ovarian cancer cells.Meanwhile,Western Blot analysis was performed to detect the effects of low and overexpression of TRIM22 protein on migration and invasion associated proteins MMP9,Vimentin,Ncadherin and Ecadherin adherin in serous ovarian cancer cells SKOV3 and A2780.Part Ⅱ: Interaction egg white of TRIM22 protein was screened by mass spectrometry in OVCAR3 cell line.The expression of TCF4,the interacting protein of TRIM22 protein,in serous ovarian cancer and normal ovarian tissue was detected by Western Blot.Co-immunoprecipitation(Co-IP)and immunofluorescence were used to detect the interaction between TRIM22 and TCF4 in OVCAR3 and SKOV3 cells of serous ovarian cancer.Immunohistochemistry and Western Blot were used to detect the correlation between TRIM22 protein and TCF4 protein expression in serous ovarian cancer.The effects of overexpression of TRIM22 and simultaneous overexpression of TRIM22+TCF4 on proliferation of serous ovarian cancer cells A2780 were detected by CCK-8 and clonal formation assay.Simultaneous Western Blot analysis of overexpression of TRIM22 protein and overexpression of TRIM22+TCF4 protein in serous ovarian cancer cells A2780 and low expression of TRIM22 protein and simultaneously low expression of TRIM22 protein and overexpression of TCF4 protein in serous ovarian cancer cells OVCAR3 pair of proliferation-related proteins PCNA and Cyclin Effect of D1 and Cyclin E.Transwell and scratch tests were used to detect the effects of overexpression of TRIM22 and simultaneous overexpression of TRIM22+TCF4 on the migration and invasion of serous ovarian cancer cells A2780.Simultaneous Western Blot analysis of overexpression and simultaneous overexpression of TRIM22 protein and TRIM22+TCF4 protein in serous ovarian cancer cells A2780 and low expression of TRIM22 protein and simultaneous low expression of TRIM22 protein and overexpression of TCF4 protein in serous ovarian cancer cells OVCAR3 on migration and invasion related proteins Vimentin,Influence of N-cadherin and E-cadherin.Part Ⅲ: CHX tracer assay was used to detect the effect of low expression of TRIM22 protein on TCF4 protein expression in serous ovarian cancer cells OVCAR3.At the same time,the effect of overexpression of TRIM22 protein on TCF4 protein expression in serous ovarian cancer cells A2780 and the effect of catalytically inactivated TRIM22/CA mutant on TCF4 protein expression in serous ovarian cancer cells A2780 were examined.Western Blot assay was used to detect the effect of low expression of TRIM22 protein and MG132 treatment on TCF4 expression in OVCAR3 serous ovarian cancer cells.The effect of low TRIM22 mRNA expression on TCF4 mRNA expression in serous ovarian cancer cells OVCAR3 was detected by Rt-PCR.HEK293 T cells were co-transfected with Flag-TRIM22 plasmid,HA tagged UB plasmid and Myc-TCF4 plasmid.Co-IP assay was used to detect the regulation of ubiquitination level of TCF4 protein by exogenous TRIM22 protein.Underexpression of TRIM22 and overexpression of TRIM22 in serous ovarian cancer cells OVCAR3 and A2780 were used to detect the regulation of endogenous TRIM22 on the ubiquitination level of TCF4 protein by Co-IP assay.Flag-TRIM22,Flag-TRIM22 C15/18 A and GST-TCF4 were synthesized by in vitro transcription and translation of proteins,and ubiquitin reaction system was performed in Escherichia coli in vitro to detect whether TRIM22 ubiquidized TCF4 in vitro.In HEK293 T cells,His-UB-K6 R,His-UB-K11 R,His-UB-K27 R,His-UB-K29 R,His-UB-K33 R,His-UB-K48 R,His-UB-K63 R,His-UB-WT,Flag-TRIM22 were co-transfected with MYC-TCF4 plasmid and treated with MG132.Co-IP assay with Myc antibody was performed to detect the ubiquitination modification type of TCF4.HEK293 T cells were cotransfected with HA-UB plasmid,Flag RING missing TRIM22 fragment,Flag B-box missing TRIM22 fragment,Flag SPRY missing TRIM22 fragment and the Coiled coil missing TRIM22 fragment of Flag.CO-IP assay with TCF4 antibody was performed to detect the domain of ubiquitination modification of TCF4 mediated by TRIM22.Results:Part Ⅰ:Gepia database showed that the expression of TRIM22 mRNA in serous ovarian cancer tissues was significantly lower than that in normal ovarian tissues(P<0.05).RT-PCR results showed that the expression of TRIM22 mRNA in 40 cases with serous ovarian cancer was 0.9834±1.0036,which was significantly lower than that in 40 cases with normal ovarian tissue(2.7560 ± 3.0701)(P<0.01).Immunohistochemical results showed that TRIM22 protein was mainly located in the nucleus,and its high expression rate in 40 serous ovarian cancer tissues and 40 normal ovarian tissues was30% and 85%,respectively(P<0.001).Western Blot results showed that TRIM22 protein expression decreased in 40 serous ovarian cancer tissues compared with 40 normal ovarian tissues(P<0.05).The clinicopathological and biological characteristics of 40 patients with serous ovarian cancer were analyzed,and TRIM22 protein was detected by immunohistochemical method.The results showed that TRIM22 protein expression was correlated with age(P<0.05),menopause(P<0.05),and lymph node metastasis(P<0.001).By analyzing the survival prognosis of 40 patients with serous ovarian cancer,the results showed that the overall survival rate of patients with serous ovarian cancer with high expression of TRIM22 protein was significantly higher than that of the group with low expression of TRIM22 protein(P=0.033).RT-PCR results showed that the expression of TRIM22 mRNA in OVCAR3,SKOV3,SW626 and HO-8910 was significantly higher than that in A2780 and Co C1(P<0.05).Western Blot results showed that the expression of TRIM22 protein in OVCAR3,SKOV3,SW626 and HO-8910 was significantly higher than that in A2780 and Co C1(P<0.05).The results of CCK-8 and clonal formation experiments showed that SKOV-3 and OVCAR3 cells with low expression of TRIM22 had enhanced proliferation ability.Western Blot results showed that SKOV-3 cells with low expression of TRIM22 protein had increased expressions of proliferation-related PCNA,Cyclin D1 and Cyclin E proteins.However,the proliferation ability of A2780 cells overexpressing TRIM22 was decreased.Western Blot results showed that the expressions of proliferation-related PCNA,Cyclin D1 and Cyclin E proteins in A2780 cells overexpressing TRIM22 protein were decreased(P<0.001).Transwell and scratch experiments showed that SKOV-3 and OVCAR3 cells with low expression of TRIM22 had enhanced invasion and migration ability.Western Blot results showed that the expressions of MMP9,Vimentin and Ncadherin were increased in SKOV-3 cells with low TRIM22 expression,while the expressions of Ecadherin were decreased.The invasion and migration ability of A2780 cells overexpressed with TRIM22 were weakened,and Western Blot results showed that the expressions of MMP9,Vimentin and Ncadherin were decreased and the expressions of Ecadherin were increased in A2780 cells overexpressed with TRIM22(P<0.001).Part Ⅱ: Mass spectrometry results showed that TCF4 had a high score,suggesting that this protein may be a key protein interacting with TRIM22 in serous ovarian cancer cells.Western Blot results showed that the expression level of TCF4 protein increased in40 serous ovarian cancer tissues compared with 40 normal ovarian tissues(P<0.05).Co-immunoprecipitation(Co-IP)results showed that the expression of TRIM22 protein was detected in the immunoprecipitation complex of TCF4,and the expression of TCF4 protein was also detected in the immunoprecipitation complex of TRIM22.Immunofluorescence results showed that TRIM22 protein and TCF4 protein overlapped in the spatial localization of cell substructure,mainly colocalized in the nucleus.Immunohistochemical results showed that TRIM22 protein was negatively correlated with TCF4 protein expression in 40 patients with serous ovarian cancer(r=-0.326,P<0.001).Western Blot results also showed a negative correlation between TRIM22 protein and TCF4 protein expression in 40 patients with serous ovarian cancer(r=-0.6201,P<0.001).The results of CCK-8 and clonal formation showed that there was no difference in the growth rate of cells overexpressing TRIM22 and simultaneously overexpressing TCF4+TRIM22,and overexpression of TCF4 could reverse the inhibition of cell proliferation caused by overexpression of TRIM22(P<0.001).Western Blot results showed that overexpression of TCF4 protein could significantly alleviate the decrease of PCNA,Cyclin D1 and Cyclin E proteins induced by overexpression of TRIM22 protein(P<0.001).Western Blot results showed that overexpression of TCF4 protein could significantly alleviate the increase of PCNA,Cyclin D1 and Cyclin E proteins with low expression of TRIM22 protein(P<0.001).The results of Transwell and scratch tests showed that there was no difference in the growth rate of cells overexpressing TRIM22 and simultaneously overexpressing TCF4+TRIM22.Overexpression of TCF4 could reverse the inhibition of cell migration and invasion induced by overexpression of TRIM22(P<0.001).Western Blot results showed that overexpression of TCF4 significantly alleviated the decrease of Vimentin and Ncadherin proteins and the increase of Ecadherin proteins detected by overexpression of TRIM22(P<0.001).Western Blot results showed that overexpression of TCF4 protein significantly alleviated the increase of Vimentin and Ncadherin protein detected by underexpression of TRIM22,and decreased the adherin protein detected by Ecadherin protein adherin(P<0.001).Part Ⅲ: The results of CHX tracer experiment showed that the low expression of TRIM22 could up-regulate the expression level of TCF4 protein and prolong the half-life of TCF4 protein.Meanwhile,overexpression of TRIM22 in serous ovarian cancer cells A2780 can down-regulate the expression level of TCF4 protein and shorten the half-life of TCF4 protein(P<0.001).Catalytic inactivation of TRIM22/CA mutants in serous ovarian cancer cells A2780 has no effect on TCF4 protein expression.Western Blot results showed that the expression of TCF4 protein in the group with low expression of TRIM22 increased after MG132 treatment(P<0.001).RT-PCR results showed that TCF4 mRNA expression was not affected by TRIM22 mRNA expression.Co-IP results showed that TCF4 binding to UB significantly increased after increasing TRIM22 protein expression.The results of endogenous Co-IP showed that the low expression of TRIM22 in OVCAR3 cells resulted in a significant decrease in TCF4 ubiquitination level.Overexpression of TRIM22 in A2780 cells led to a significant increase in TCF4 ubiquitination.The results of in vitro ubiquitination experiments showed that TRIM22 could ubiquitinate TCF4 in vitro.Co-IP experiment showed that TRIM22 could transfer K48 ubiquitin chain to TCF4 in HEK293 T cells.TRIM22 without RING domain cannot be combined with TCF4.TCF4 without the DBD and AD domains cannot be combined with TRIM22.Conclusion: 1.TRIM22 mRNA and protein are low expressed in serous ovarian cancer tissues,and the expression level of TRIM22 protein is related to the clinicopathologic characteristics of serous ovarian cancer patients,including age,menopause and lymph node metastasis.Patients with serous ovarian cancer with low expression of TRIM22 protein have poor survival prognosis.2.The study on the influence of TRIM22 on the malignant biological behavior of serous ovarian cancer shows that TRIM22 plays a cancer-suppressing role in serous ovarian cancer and participates in the occurrence and development of ovarian cancer.3.TRIM22 protein can affect the proliferation,invasion and migration of serous ovarian cancer cells by regulating TCF4 protein.4.TRIM22 mediated the K48 ubiquitination modification of TCF4 through the RING domain,promoting the development of serous ovarian cancer. |
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