Functional investigation of miRNAs by characterization of SH-SY5Y cells overexpressing wild type or mutant miRNA genes

Genome wide mRNA and miRNA profiling was performed in SH-SY5Y cells stably overexpressing wild type or mutant MIR204 or MIR618. Mutants came from a large scale genetic screening of brain expressed miRNA genes in patients with schizophrenia or idiopathic generalized epilepsy and in control individuals. Based on enrichment of the variants with the schizophrenic or epileptic phenotype and based on impact prediction, two variants, one near MIR204 (rs7861254) and one in MIR618 (rs2682818) were selected for functional validation. Genome wide profiling of mRNA (micro-array) and mature miRNAs (small RNA sequencing, submitted to SRA) was performed in the created stable cells to assess the effect of the variants and to investigate the function of these miRNA genes. Micro-array analysis on HTA2.0 array was performed at AROS Applied Biotechnology, Denmark. SH-SY5Y cells (purchased from Sigma-Aldrich, catalog number: 94030304-1VL) were transduced with MIR204 or MIR618 wild type or mutant constructs. The stable cells were then split to obtain biological replicates and were cultured separately prior to total RNA extraction and micro-array analysis on the HTA2.0 array. For MIR204 analysis: 2 wt cells versus 3 mutant cells; for MIR618 analysis: 3 wt cells versus 3 mutant cells were used.