ABSTRACT
BACKGROUND: Ankyrin repeat and SOCS box protein 3 (ASB3) is a member of ASB family and contains ankyrin repeat sequence and SOCS box domain. Previous studies indicated that it mediates the ubiquitination and degradation of tumor necrosis factor receptor 2 and is likely involved in inflammatory responses. However, its effects on oncogenesis are unclear. This study aimed to investigate the effects of ASB3 on the growth and metastasis of colorectal cancer (CRC). METHODS: We used next-generation sequencing or Sanger sequencing to detect ASB3 mutations in CRC specimens or cell lines, and used real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical or immunofluorescence assay to determine gene expression. We evaluated cell proliferation by MTT and colony formation assays, tested cell cycle distribution by flow cytometry, and assessed cell migration and invasion by transwell and wound healing assays. We also performed nude mouse experiments to evaluate tumorigenicity and hepatic metastasis potential of tumor cells. RESULTS: We found that ASB3 gene was frequently mutated (5.3%) and down-regulated (70.4%) in CRC cases. Knockdown of endogenous ASB3 expression promoted CRC cell proliferation, migration, and invasion in vitro and facilitated tumorigenicity and hepatic metastasis in vivo. Conversely, the ectopic overexpression of wild-type ASB3, but not that of ASB3 mutants that occurred in clinical CRC tissues, inhibited tumor growth and metastasis. Further analysis showed that ASB3 inhibited CRC metastasis likely by retarding epithelial-mesenchymal transition, which was characterized by the up-regulation of ß-catenin and E-cadherin and the down-regulation of transcription factor 8, N-cadherin, and vimentin. CONCLUSION: ASB3 dysfunction resulted from gene mutations or down-regulated expression frequently exists in CRC and likely plays a key role in the pathogenesis and progression of CRC.
Subject(s)
Colorectal Neoplasms , Suppressor of Cytokine Signaling Proteins/genetics , Animals , Asian People/genetics , Cell Cycle , Cell Line , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred BALB C , Mutation , Suppressor of Cytokine Signaling Proteins/metabolism , Wound HealingABSTRACT
In this study, we tried to explore if xeroderma pigmentosum complementation group-A (XPA) expression is likely a prognostic prediction factor for locally advanced nasopharyngeal carcinoma (NPC) patients treated with platinum-based chemoradiotherapy, which was considered to bring chemotherapy-related severe toxicity compared with radiotherapy alone. Firstly, MTT assay revealed that downregulating XPA expression in NPC HONE1 and CNE1 cells decreased IC50 of cisplatin and sensitized cells to cisplatin. XPA expression was detected by immunohistochemistry in cancer tissues from locally advanced NPC patients treated with platinum-based chemoradiotherapy. The relationships between XPA expression and clinicopathologic features, overall survival and progression-free survival of patients were evaluated. The results showed that XPA expression was not associated with clinicopathologic parameters, but was likely an independent prognostic factor for patient survival. High XPA level predicts a poor prognosis, and the prediction values were higher in subgroups of younger, higher EBV antibody titer, or treated with concurrent chemoradiotherapy. Combining XPA levels and T/N classifications, we successfully classified these patients into low, medium and high risk groups for platinum-based chemoradiotherapy. These findings suggest that XPA levels may be a potential predictor of prognosis in locally advanced NPC patients treated with platinum-based chemoradiotherapy, and helpful for selecting patients likely to need and benefit from this treatment in future.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/therapy , Xeroderma Pigmentosum Group A Protein/metabolism , Blotting, Western , Carcinoma , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Chemoradiotherapy/methods , Cisplatin/administration & dosage , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Immunohistochemistry , Male , Middle Aged , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/genetics , Outcome Assessment, Health Care/methods , Outcome Assessment, Health Care/statistics & numerical data , Prognosis , Proportional Hazards Models , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Xeroderma Pigmentosum Group A Protein/geneticsABSTRACT
OBJECTIVE: To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica. METHODS: The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. RESULTS: The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult. CONCLUSION: The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.