ABSTRACT
Genetic association between E3 ubiquitin ligase SMURF2 and colorectal cancer (CRC) has been identified, while the mechanism remains undefined. Tumor-promoting gene YY1 represents a downstream factor of SMURF2. The study was designed to evaluate the effect of SMURF2 on the malignant phenotypes of CRC cells and the underlying mechanism. The expression pattern of SMURF2 and YY1 in CRC clinical tissues and cells was characterized by immunohistochemistry (IHC) and Western blot. Gain- and loss-of-function experiments were conducted to assess the effect of SMURF2 and YY1 on the behaviors of CRC cells. After bioinformatics analysis, the relationship between YY1 and SENP1 as well as between SENP1 and c-myc was determined by luciferase reporter and ChIP assays. Rescue experiments were performed to show their involvement during CRC progression. Finally, in vivo models of tumor growth were established for validation. SMURF2 was lowly expressed and YY1 was highly expressed in CRC tissues and cells. YY1 overexpression resulted in promotion of CRC cell proliferation, migration, and invasion, which could be reversed by SMURF2. Furthermore, SMURF2 could induce ubiquitination-mediated degradation of YY1, which bound to the SENP1 promoter and upregulated SENP1 expression, leading to enhancement of c-myc expression. The in vivo data revealed the suppressive role of SMURF2 gain-of-function in tumor growth through downregulation of YY1, SENP1, or c-myc. Altogether, our data demonstrate the antitumor activity of SMURF2 in CRC and the anti-tumor mechanism associated with degradation of YY1 and downregulation of SENP1/c-myc.
Subject(s)
Colorectal Neoplasms , Ubiquitin-Protein Ligases , Humans , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Cell Proliferation/genetics , Down-Regulation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , YY1 Transcription Factor/genetics , YY1 Transcription Factor/metabolismABSTRACT
Colorectal carcinoma (CRC) poses heavy burden to human health and has an increasing incidence. Currently, the existing biomarkers for CRC bring about restrained clinical benefits. GSK3ß is reported to be a novel therapeutic target for this disease but with undefined molecular mechanisms. Thus, we aimed to investigate the regulatory effect of GSK3ß on CRC progression via FTO/MZF1/c-Myc axis. Firstly, the expression patterns of GSK3ß, FTO, MZF1 and c-Myc were determined after sample collection. Lowly expressed GSK3ß but highly expressed FTO, MZF1 and c-Myc were found in CRC. After transfection of different overexpressed and interference plasmids, the underlying mechanisms concerning GSK3ß in CRC cell functions were analysed. Additionally, the effect of GSK3ß on FTO protein stability was assessed followed by detection of MZF1 m6A modification and MZF1-FTO interaction. Mechanistically, GSK3ß mediated ubiquitination of demethylase FTO to reduce FTO expression. Besides, GSK3ß inhibited MZF1 expression by mediating FTO-regulated m6A modification of MZF1 and then decreased the proto-oncogene c-Myc expression, thus hampering CRC cell proliferation. We also carried out in vivo experiment to verify the regulatory effect of GSK3ß on CRC via FTO-mediated MZF1/c-Myc axis. It was found that GSK3ß inhibited CRC growth in vivo which was reversed by overexpressing c-Myc. Taken together, our findings indicate that GSK3ß suppresses the progression of CRC through FTO-regulated MZF1/c-Myc axis, shedding light onto a new possible pathway by which GSK3ß regulates CRC.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Colorectal Neoplasms/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Kruppel-Like Transcription Factors/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Animals , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Mice , Models, Biological , Proto-Oncogene Mas , Xenograft Model Antitumor AssaysABSTRACT
Cancer cell-derived extracellular vesicles (EVs) have been reported to promote the progression of colorectal cancer (CRC), although the regulatory mechanism remains uncharacterized. In this study, we investigated the role of microRNA-25 (miR-25)/sirtuin 6 (SIRT6) in the contribution of EVs derived from CRC cells to progression of CRC. In a co-culture system with EVs from HCT116 and NCM460 cells, the viability, migratory, and invasive properties of SW480 and SW620 cells were evaluated by cell counting kit-8 (CCK-8) and Transwell assays. Luciferase, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation (RIP) assays were conducted to verify the interaction among miR-25, SIRT6, lin-28 homologB (Lin28b), and neuropilin-1 (NRP-1). It was established that HCT116 cell-derived EVs promoted the malignant properties of SW480 cells and SW620 cells by delivering miR-25. SIRT6 was targeted by miR-25, whereas SIRT6 inhibited NRP-1 through downregulation of Lin28b. The tumor-bearing nude mouse experiments substantiated that HCT116 cell-derived EVs transferred miR-25 to facilitate tumor formation and metastasis by inhibiting SIRT6. In summary, our study clarifies the involvement of miR-25-targeted SIRT6 inhibition and SIRT6-mediated inhibition of the Lin28b/NRP-1 axis in CRC cell-derived EVs to CRC progression and metastasis.
