TRIM29 Regulates The SETPB1/SET/PP2A Axis Via Transcription Factor VEZF1 To Promote Progression Of Ovarian Cancer

Objective: Ovarian cancer is one of the common malignant tumors derived from the female reproductive system.Moreover,it is posing a severe threat to the health and life of women worldwide due to its mortality which has ranked the first among all types of gynecological tumors.With the development of molecular medicine and tumor-targeted therapy,the clinical effects of ovarian cancer have been greatly improved.However,the overall prognosis of patients with ovarian cancer remains relatively poor.Since tumor development is a complex biological process accompanied by abnormal expression of a wide range of genes,it is of great clinical significance to investigate the potential mechanisms underlying the progression of ovarian cancer to identify critical molecular targets for developing new treatments that may work better.TRIM29 is a member of the tripartite motif-containing protein(TRIM)family.Unlike most TRIM family proteins with classic three-domain features,no RING finger domain is contained in the TRIM29 protein.But the N-terminal of TRIM29 consists of two zinc finger domains(B-box1 and B-box2)and a coiled-coil domain.As a classic member of the TRIM protein family,TRIM29 has been found to be implicated in various biological processes,including transcriptional regulation,morphological development,cell proliferation,cell differentiation and autophagy.In recent years,growing evidence has revealed an essential role of TRIM29 in the occurrence and progression of tumors.However,previous reports on the specific functions of TRIM29 in different types of tumors are not always consistent.Therefore,whether TRIM29 acts as a potential tumor-promoting factor or a tumor-suppressing one should be further elucidated in the genetic context of a particular type of tumor.SETBP1 is a recognized oncoprotein that protects SET,an endogenous inhibitor of protein phosphatase 2A(PP2A),from proteasome degradation and thereby stabilizes the full-length SET.SET,also known as inhibitor-2 of protein phosphatase 2A(I2PP2A),inactivates PP2 A via the recruitment of kinases that phosphorylate PP2 A at tyrosine 307.While the unphosphorylated PP2 A functions as a classic tumor suppressor by targeting multiple kinases related to proliferation,cell cycle and apoptosis in most types of tumors.Our previous work has demonstrated that TRIM29 was significantly upregulated in most types of ovarian cancers,including the serous,mucinous and clear cell ovarian cancer,which contributed to the maintenance of the cancer stem cell-like(CSC-like)phenotype in ovarian cancer cells.To this end,the present study aimed to 1)elucidate the regulatory effect of TRIM29 on the SETBP1/SET/PP2 A axis to reveal the underlying mechanism by which TRIM29 promotes the malignant progression of ovarian cancer;2)provide scientific clues and a theoretical foundation for the identification of promising therapeutic targets for ovarian cancer.Methods: 1.Ovarian cancer cell lines with TRIM29 knockout were constructed using the CRISPR/Cas9 system.2.A global proteomic analysis was conducted to identify potential molecules regulated by TRIM29 in ovarian cancer cells.3.The effects of TRIM29 knockdown on the SETBP1/SET/PP2 A axis in CAOV3 and A2870 ovarian cancer cells were detected by Western blot.4.The correlation between TRIM29 and SETBP1 expression in ovarian cancer was evaluated by immunohistochemical and bioinformatic analyses.5.Ovarian cancer cells with TRIM29 knockdown were treated with a PP2 A inhibitor LB100 to detect the independent viability,invasiveness and spheroid formation efficiency of cells,respectively evaluated by the colony formation assay,Transwell invasion assay and spheroid formation assay.6.SETBP1 overexpression was conducted using the way of lentiviral infection to detect the effects of the recovery of SETBP1 expression on the independent viability,invasiveness,spheroid formation efficiency and tumorigenicity in nude mice of ovarian cancer cells with TRIM29 knockdown.7.Total and newly-synthesized m RNA levels of SETBP1 were measured by RT-q PCR in ovarian cancer cells with TRIM29 knockdown to detect the effects of TRIM29 on the transcription of the SETBP1 gene.8.The luciferase reporter gene plasmid containing the SETBP1 promoter sequences was constructed and then transfected into cells for 48 h to detect the effects of TRIM29 knockdown on SETBP1 promoter activity.9.Immunofluorescence and nucleus-cytoplasm separation assays were performed to detect the distribution of TRIM29 in ovarian cancer cells.10.Bioinformatic analysis was conducted to evaluate the correlation between SETBP1 and VEZF1 expression in ovarian cancer and screen potential binding sites of VEZF1 on the SETBP1 promoter.11.The dual-luciferase reporter gene assay and Ch IP assay were performed to respectively detect the effects of VEZF1 on SETBP1 promoter activity and the direct interaction between VEZF1 and SETBP1 promoter.12.The sh RNA targeting VEZF1 was transfected into control or TRIM29 knockdown CAOV3 and A2780 ovarian cancer cells to silence the expression of VEZF1.13.The effects of VEZF1 knockdown on the independent viability,invasiveness and spheroid formation efficiency of ovarian cancer cells were evaluated by the colony formation assay,Transwell invasion assay and spheroid formation assay.14.VEZF1 m RNA levels were measured by RT-q PCR to evaluate the effects of TRIM29 knockdown on the transcription of the VEZF1 gene in ovarian cancer cells.15.The control or TRIM29 knockdown ovarian cancer cells were respectively treated with the protein synthesis inhibitor cycloheximide(CHX),proteasome inhibitor MG132 and lysosomal inhibitor E64D+pepstain A to clarify the effects of TRIM29 on the synthesis or degradation of VEZF1 protein.16.The luciferase reporter gene plasmids containing the VEZF1 m RNA 5’UTR or 3’UTR were constructed and then transfected into cells for 48 h to evaluate the effects of TRIM29 on the activity of VEZF1 m RNA 5’UTR or 3’UTR.17.Mass spectrometry was conducted to screen potential RNA-binding proteins(RBPs)that bind to the VEZF1 m RNA 3’UTR.18.RNA immunoprecipitation(RIP)and pull-down were performed to confirm the candidate RBPs that bind to VEZF1 m RNA 3’UTR in ovarian cancer cells.19.Bioinformatic analysis was conducted to predict potential binding sites of candidate RBPs on the VEZF1 m RNA 3’UTR.20.The luciferase reporter gene plasmids containing partial deletion mutants of VEZF1 m RNA 3’UTR were constructed and then transfected into cells for 48 h to confirm the required sequences for the candidate RBPs to bind to the VEZF1 m RNA 3’UTR.21.TRIM29 was knocked down in CAOV3 and A2780 ovarian cancer cells to evaluate the effects of TRIM29 knockdown on the binding of candidate RBPs to the VEZF1 m RNA 3’UTR.Results: 1.SETBP1 was identified as the key molecular target regulated by TRIM29 in ovarian cancer cells.2.A significantly positive correlation between TRIM29 and SETBP1 expression was found in ovarian cancer tissues.3.A high expression level of TRIM29 or SETBP1 indicated an undesirable prognosis for patients with ovarian cancer.4.The suppression of CSC-like features in ovarian cancer by TRIM29 knockdown mainly depended on the inactivation of the SETBP1/SET/PP2 A axis.5.TRIM29 knockdown inhibited the transcriptional activity of the SETBP1 promoter in ovarian cancer cells.6.TRIM29 was not a transcription factor for SETBP1 since it was almost entirely localized in the cytoplasm.7.TRIM29 regulated the transcriptional activity of SETBP1 via a transcription factor VEZF1 in ovarian cancer cells.8.VEZF1 knockdown impaired the independent viability,invasiveness and spheroid formation efficiency of ovarian cancer cells,which was lapsed by TRIM29 knockdown.9.TRIM29 knockdown had no impact on the m RNA levels of VEZF1 but decreased its protein levels.10.TRIM29 promoted the translation of VEZF1 m RNA in a 3’UTR-dependent manner.11.BICC1,an RBP binding to the 3’UTR of VEZF1 m RNA,facilitated VEZF1 m RNA translation.12.TRIM29 knockdown decreased VEZF1 protein levels via weakening the binding of BICC1 to the VEZF1 m RNA 3’UTR.Conclusion: TRIM29 drives the malignant progression of ovarian cancer via regulating the SETBP1/SET/PP2 A oncogenic signal axis.Mechanistically,TRIM29 promoted VEZF1 m RNA translation by recruiting the BICC1 to interact with its 3’UTR,which therefore facilitated the transcriptional activation of SETBP1 mediated by VEZF1.Consequently,the continuously activated SETBP1/SET/PP2 A axis maintained the CSC-like phenotype of ovarian cancer cells.In a word,TRIM29 might be a potential biomarker for the prognosis of ovarian cancer and a possible therapeutic target for ovarian cancer.