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

Gonadotroph neuroendocrine pituitary tumors are among the most common intracranial neoplasms. A notable proportion of these tumors is characterized by invasive growth which hamper the treatment results and worsen prognosis for patients. Increased hsa-miR-184 expression was observed in invasive gonadotroph tumor. This study was aimed to determine the role 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 in tumor 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 used for transcriptome analysis in mir-184 treated and untreated LβT2 cells. Differential expression analysis combined with target prediction were used to identify miR-184 target followed by its validation with Luciferase reporter assay.

Analysis of tissue samples showed that hsa-miR-184 is downregulated in gonadotroph tumors and its expression is lower in invasive that 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 increasing cellular proliferation and viability. Differentially expressed genes were identified when comparing miR-184 treated and untreated cells including only Nus1 as predicted miR-184 target. The interaction between miR-184 and 3’UTR of Nus1 was confirmed in both LβT2 and αT3-1. Overexpression of Nus1 resulted in lowering cell viability in both cells and proliferation in LβT2. The expression level of NUS1 was found lowered in invasive than noninvasive tumors.

Our results indicate that DNA hypomethylation-related increase of hsa-mir-184 expression contribute to invasive growth of gonadotroph pituitary tumors through targeting NUS1- being one the various molecular mechanisms involved acquiring the aggressive growth potential..fastq.gz files are raw sequences, which were aligned to GRCm39 genome by STAR aligner, subsequently differentiall expression with DESeq2 was performed (DEGs_mir184.csv) and gene set expression analysis was accomplished with fgsea against a range of gene sets (Reactome, Gene Ontology, KEGG, etc.); these results are stored in gsea.zip.