Reprimo (RPRM), a downstream effector of p53-induced cell cycle arrest at G2/M, has been proposed as a putative tumor suppressor gene (TSG) and as a potential biomarker for non-invasive detection of gastric cancer (GC). The aim of this study was to evaluate the epigenetic silencing of RPRM gene by promoter methylation and its tumor suppressor function in GC cell lines. Furthermore, clinical significance of RPRM protein product and its association with p53/p73 tumor suppressor protein family was explored. Epigenetic silencing of RPRM gene by promoter methylation was evaluated in four GC cell lines. Protein expression of RPRM was evaluated in 20 tumor and non-tumor matched cases. The clinical significance of RPRM association with p53/p73 tumor suppressor protein family was assessed in 114 GC cases. Tumor suppressor function was examined through functional assays. RPRM gene expression was negatively correlated with promoter methylation (Spearman rank r = -1; p = 0.042). RPRM overexpression inhibited colony formation and anchorage-independent growth. In clinical samples, RPRM gene protein expression was detected in 75% (15/20) of non-tumor adjacent mucosa, but only in 25% (5/20) of gastric tumor tissues (p = 0.001). Clinicopathological correlations of loss of RPRM expression were significantly associated with invasive stage of GC (stage I to II-IV, p = 0.02) and a positive association between RPRM and p73 gene protein product expression was found (p<0.0001 and kappa value = 0.363). In conclusion, epigenetic silencing of RPRM gene by promoter methylation is associated with loss of RPRM expression. Functional assays suggest that RPRM behaves as a TSG. Loss of expression of RPRM gene protein product is associated with the invasive stage of GC. Positive association between RPRM and p73 expression suggest that other members of the p53 gene family may participate in the regulation of RPRM expression.

Reprimo-like (RPRML) is an uncharacterized member of the Reprimo gene family. Here, we evaluated the role of RPRML and whether its regulation by DNA methylation is a potential non-invasive biomarker of gastric cancer. RPRML expression was evaluated by immunohistochemistry in 90 patients with gastric cancer and associated with clinicopathologic characteristics and outcomes. The role of RPRML in cancer biology was investigated in vitro, through RPRML ectopic overexpression. Functional experiments included colony formation, soft agar, MTS, and Ki67 immunofluorescence assays. DNA methylation-mediated silencing was evaluated by the 5-azacytidine assay and direct bisulfite sequencing. Non-invasive detection of circulating methylated RPRML DNA was assessed in 25 gastric cancer cases and 25 age- and sex-balanced cancer-free controls by the MethyLight assay. Downregulation of RPRML protein expression was associated with poor overall survival in advanced gastric cancer. RPRML overexpression significantly inhibited clonogenic capacity, anchorage-independent growth, and proliferation in vitro. Circulating methylated RPRML DNA distinguished patients with gastric cancer from controls with an area under the curve of 0.726. The in vitro overexpression results and the poor patient survival associated with lower RPRML levels suggest that RPRML plays a tumor-suppressive role in the stomach. Circulating methylated RPRML DNA may serve as a biomarker for the non-invasive detection of gastric cancer.

15 Background: Gastric cancer (GC) is the third leading cause of cancer death worldwide. Chronic infection by H.pylori and EBV is related to the multistep cascade of GC. This cascade hypothesizes that a specific sequence of lesions leads to GC. Epigenetic processes, such as DNA methylation or miRNAs, are new players in the pathogenesis of GC. We aim to identify novel upregulated microRNAs associated with the multistep cascade through the use of open access databases and bioinformatics tools. Methods: We selected 3 GEO DataSets with human miRNA expression levels in paired tumor and non-tumor samples from gastric cancer patients. The largest library, GSE 23739 (40 matched T/N samples) was used as a "discovery dataset”. Only miRNAs upregulated >1.2 fold T over N were considered. Upregulated miRNAs were further validated in two independent datasets: i) GSE28700 (22 fresh-frozen paired T and N samples) and ii) GSE54397 (16 formalin-fixed T and N samples) and confirmed with TCGA Stomach Adenocarcinoma (STAD) data (doi:10.1038/nature13480). Finally, upregulated miRNAs were analyzed by Diana Tools miRPath, for associated oncogenic pathways and target genes. Results: From the discovery dataset GSE 23739 we identified 231 upregulated miRNAs between 40 matched T/N samples. Only 12 of these candidates were found in both validation datasets GSE28700 and GSE54397. Only three of these miRNAs were undescribed in GC: miRNAs-505, -552 and -592. TCGA STAD data confirmed our results. Analysis in miRPath revealed association with three oncogenic pathways PI3K-AKT, TGFβ and Erbβ; as well as genes relevant in oncogenesis: MAP2K1, RPS6KB1, TGFA, SMAD3, IGF1, LAMC1, IFNAR1, ITGB6, PIK3CA, PPP2R2D, EIF4E, BRCA1, FOXO3 and CREB3L3. Conclusions: The use of open access databases and bioinformatics tools enabled us to identify three novel upregulated microRNAs with oncogenic potential in GC. The analysis of these miRNAs revealed associated oncogenic pathways and target genes. These findings need to be further inquired in an experimental manner in order to assess the potential role of these miRNAs in the multistep cascade of GC.