The expression of legumain which has been shown overexpressed in patients with metastatic gastric cancer is positively correlated to both disease progression and outcome, and negatively correlated to microRNA (miR)−3978 expression. The RNA-binding protein, poly r(C) binding protein 1 (PCBP1) was the most downregulated protein in the metastatic tissue specimens. Quantitative real-time PCR showed that PCBP1 expression is transcriptionally downregulated in peritoneal metastasis tissues. RNA immunoprecipitation experiments showed that PCBP1 and miR-3978 are sequestered in normal peritoneal tissue, but the complex is disrupted following metastatic progression. PCBP1 expression mimicked miR-3978 expression across gastric cancer patients. Finally, replenishment of PCBP1 or miR-3978 expression in the peritoneal metastasis cell line MKN45 decreased legumain protein expression and chemosensitized the cells to treatment with docetaxel. However, replenishment of one and concomitant depletion of the other failed to induce chemosensitivity to docetaxel. Replenishment of miR-3978 also resulted in induction of PCBP1 protein expression, potentially indicating that miR-3978 expression might downregulate a negative regulator targeting PCBP1. Our current study reveals PCBP1 as an additional biomarker in peritoneal metastasis. PCBP1 and miR-3978 expression were correlated and suggests a potential interplay of differential miRNA biogenesis and RNA binding protein during metastatic progression.
In China, majority of the mortality in gastric cancer are associated with peritoneal metastasis. Since most gastric tumors are metastatic at initial diagnosis, the treatment of gastric cancer is limited to radical resection. Therefore, it is imperative to identify diagnostic and prognostic biomarkers. From 2014 to 2015, 20 patients were enrolled in the study. To search translationally upregulated genes in the context of epithelial to mesenchymal transition (EMT), polysome profiling was performed. The MTT, migration, and invasion assay were conducted to determine cell proliferation, migration, and invasion ability respectively. Experiments of gain and loss of function were performed using the overexpression plasmid, siRNA, and shRNA. Xenograft assay was established using nude mice to explore the role of targets translationally upregulated gene in vivo. Polysome profiling defined the landscape of translationally regulated gene products with differential expression between non-metastatic and metastatic cohorts. Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) was found to be the most translationally upregulated gene product in either experimental groups. STEAP1 was found to be required for cell proliferation, in vitro migration and invasion, and in vivo tumorigenesis. RNAi-mediated silencing of STEAP1 potentiated chemosensitivity of the MKN45 cells to docetaxel treatment, highlighting the importance of STEAP1 as a novel biomarker in gastric cancer patients with peritoneal metastasis. STEAP1 is thus induced translationally and its expression promotes proliferation, migration, invasiveness, and tumorigenicity of gastric cancer. STEAP1 can be a potent candidate for designing of targeted therapy.
Gastric cancer incidence and mortality are among the highest in China, with majority of the mortality related to peritoneal metastasis of gastric cancer. Treatment is limited to radical resection, which is impeded by incidence of metastasis at time of initial diagnosis, thus making it imperative to identify diagnostic and prognostic biomarkers. Legumain, a lysosomal cysteine endopeptidase of the asparaginyl endopeptidase family, has been shown to be overexpressed in patients with metastatic gastric cancer disease and its expression was positively correlated to both disease progression and outcome. However, the mechanism of legumain expression is currently unknown. Legumain overexpression was found to occur at the level of post transcriptional gene regulation. In situ prediction algorithms identified legumain as a putative target of miR-3978. MiR-3978 was significantly decreased in peritoneal metastatic tissue specimens and in MKN45 cells that mimic peritoneal metastasis features. Reporter assays using LGMN (encoding legumain) 3′ untranslated region (UTR) showed that miR-3978 interacted with the wild-type but not miR-3978-seed mutant. Ectopic expression of miR-3978 mimic in the MKN45 cell line significantly decreased proliferation and suppressed in vitro migration and invasion. The miR-3978 mimic inhibited gastric carcinoma and metastatic progression in a mice model by regulating legumain protein expression. Inverse correlation of LGMN mRNA and miR-3978 levels in 20 gastric patients at different stages of metastatic disease confirmed the same. Cumulatively, our results indicate that loss of miR-3978 leads to increased expression of legumain, which indicates that miR-3978might be a biomarker for peritoneal metastasis in patients with gastric cancer.
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