Epigenetic dysregulations of miR-30a and LINE-1 in gastric cancer

Abstract

Background: Epigenetic alterations such as microRNA (miRNA) and DNA methylation can regulate cancer cell properties in gastric cancer. miRNAs are an abundant class of negative gene regulators that control a wide range of biological functions such as cellular proliferation, differentiation and apoptosis by regulating multiple gene targets. miR-30a has been reported as a downregulated miRNA in gastric cancer, but its biological function and clinical implication have not been reported much. DNA methylation can repress transposable elements such as long interspersed nuclear element-1 (LINE-1), and LINE-1 is frequently hypomethylated in cancer. LINE-1 retrotransposition can generate many small RNAs such as miRNAs, and LINE-1 silencing is associated with miR-30a. Purpose: The aim of this study was to identify miR-30a expression and LINE-1 methylation patterns as well as miR-30a biological function by finding its gastric cancer-specific target genes. We also checked whether both miR-30a and LINE-1 could be diagnostic or prognostic markers in gastric cancer by assessing the clinical impacts. Method: We performed qRT-PCR using our tissue samples to identify miR-30a and its target gene expressions. We used data from TCGA and NCBI GEO, to confirm their expressions in open source databases. To determine their biological functions, miR-30a was overexpressed by mimics or inhibited by inhibitors in gastric cancer cell lines. Moreover, a stable cell line overexpressing miR-30a was used for the in vivo tumorigenesis assay. Microarray was introduced to confirm the gastric cancer-specific target gene of miR-30a, which was later knocked down by siRNA for functional studies. For LINE-1 methylation analysis, we examined four CpG sites of LINE-1 by quantitative bisulfite pyrosequencing using frozen and formalin-fixed paraffin-embedded (FFPE) tissues of gastric cancer. Finally, we analyzed the clinicopathological data. Result: In gastric cancer tissues, miR-30a was down-regulated, and LINE-1 was hypomethylated when compared to normal tissues. Ectopic expression of miR-30a decreased cell growth, migration capacity and colony formation in vitro and in vivo. Furthermore, we found that miR-30a directly targeted the ITGA2 gene and that the miR-30a-ITGA2 axis was significantly related to H. pylori-infected and microsatellite instability (MSI)-high gastric cancer. Higher expression of ITGA2 was particularly exhibited in intestinal type gastric cancer than in diffuse type gastric cancer. When we used both frozen and FFPE tissues for LINE-1 methylation analysis, LINE-1 was differentially methylated between two types of tissues. In frozen tissues, LINE-1 methylation status was different according to gender, differentiation, lymphatic and venous invasions. In FFPE tissues, LINE-1 methylation was significantly different according to tumor location and venous invasion. Conclusion: Cumulatively, miR-30a functions as a tumor suppressor by directly targeting ITGA2. miR-30a-ITGA2 axis is related to several clinicopathological features of gastric cancer such as H. pylori, MSI and intestinal type. LINE-1, as one of miR-30a regulators, can be a marker according to its methylation status for several clinicopathological parameters, and especially it can be a marker for venous invasion in irrespective of gastric cancer tissue types. Therefore, we suggest that both miR-30a-ITGA2 axis and LINE-1 may be useful strategies for treatment and prediction of prognosis in gastric cancer. LINE-1 study was published in Molecules and Cells in 2017, and figures and tables were reproduced and used in this paper [1].