Experimental validation of candidate schizophrenia gene ZNF804A as target for hsa-miR-137

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that mainly function as negative regulators of gene expression (Lai, 2002) and have been shown to be involved in schizophrenia etiology through genetic and expression studies (Burmistrova et al., 2007; Hansen et al., 2007a; Perkins et al., 2007; Beveridge et al., 2010; Kim et al., 2010). In a mega analysis of genome-wide association study (GWAS) of schizophrenia (SZ) and bipolar disorders (BP), a polymorphism (rs1625579) located in the primary transcript of a miRNA gene, hsa-miR-137, was reported to be strongly associated with SZ. Four SZ loci (CACNA1C, TCF4, CSMD1, C10orf26) achieving genome-wide significance in the same study were predicted and later experimentally validated (Kwon et al., 2011) as hsa-miR-137 targets. Here, using in silico, cellular and luciferase based approaches we also provide evidence that another well replicated candidate schizophrenia gene, ZNF804A, is also target for hsa-miR-137.

Fig. 1

(A) Analysis of ZNF804A expression in HEK293 cells over expressing miR-137; (B) ZNF804A expression analysis in Be2C cells over expressing miR-137. The error bars represent the SE±95% CI from three independent experiments (***pb0.001; **pb0.01).

Fig. 2

(A) The miR-137 mature sequence is aligned with wild type and mutant ZNF804A sequences. The alignment of nucleotide seed region (2–7nt) is shown with vertical lines. Mutated nucleotides in the seed region are boxed in the red rectangular. (B) Luciferase activity were measured in the empty vector (gray column) and compared to ZNF804A WT (green bar), ZNF804A MT (red bar), ZNF804A WT with anti-miR-137 oligo (blue bar), and ZNF804A WT in presence of miR-377 construct (yellow bar). Results were generated through the nonparametric Kruskal–Wallis test and are reported as average of four replicates from three independent experiments. Error bars represents SE±95% CI; **pb0.01.