DNA hypomethylation-related expression of hsa-miR-184 contributes to invasive growth of gonadotroph neuroendocrine pituitary tumors

Abstract

Gonadotroph neuroendocrine pituitary tumors are among the most common intracranial neoplasms. A notable proportion of these tumors is characterized by invasive growth which hampers the treatment results and worsens prognoses of patients. Increased hsa-miR-184 expression was observed in invasive as compared to non-invasive gonadotroph tumors. This study aimed to determine the role of hsa-miR-184 expression in invasive growth of gonadotroph tumors. QRT-PCR and bisulfite pyrosequencing were used for evaluating hsa-miR-184 expression and MIR184 DNA methylation levels, respectively, in tumors and normal pituitary samples. LβT2 and αT3-1 gonadotroph cells were used to test the effect of miR-184 on cell viability (MTT test), proliferation (BrdU incorporation), and migration (scratch assay). RNA sequencing was applied for transcriptome profiling in miR-184-treated and untreated LβT2 cells. Differential genes expression analysis combined with target prediction served for identification of miR-184 targets. MiRNA-mRNA interaction was subsequently validated with Luciferase reporter assay. Analysis of tissue samples showed that hsa-miR-184 is upregulated in gonadotroph tumors and its expression is higher in invasive than in noninvasive ones. Promoter of MIR184 is demethylated in tumors, and the methylation level is negatively correlated with hsa-miR-184 expression. Transfecting LβT2 and αT3-1 with miR-184 mimic resulted in increased cellular proliferation and viability. Differentially expressed genes were identified when comparing miR-184-treated and untreated cells, including Nus1 as the only predicted miR-184 target. The interaction between miR-184 and 3'UTR of Nus1 was confirmed in vitro in both LβT2 and αT3-1. Overexpression of Nus1 resulted in lowering cell viability in both cell lines and proliferation in LβT2. The expression level of NUS1 was lower in invasive than in noninvasive tumors. Our results indicate that DNA hypomethylation-related increase of hsa-mir-184 expression contributes to invasive growth of gonadotroph pituitary tumors through targeting NUS1, being one of the various molecular mechanisms involved in conferring aggressive growth potential.