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

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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.

Introduction

Both SZ and BP disorders are debilitating psychiatric illnesses that pose a major burden on public health due to early onset and for many patients, the need for long-term care. While the etiology of these disorders is still unknown, in conjunction with environmental and developmental factors (Kendler et al., 1985, Kety, 1987, Jablensky et al., 1992a, Jablensky et al., 1992b, Kendler and Diehl, 1993a, Kendler and Diehl, 1993b, Kendler et al., 1994a, Kendler et al., 1994b, Kendler et al., 1996, Hansen et al., 2007b) there is consistent evidence for a substantial genetic component (Sullivan et al., 2003, Kato et al., 2005) (heritability ~ 80%) with some shared between both diseases (Berrettini, 2003, Purcell et al., 2009a).

GWAS are a powerful, systematic and unbiased genetic approach to study the common disease/common variant (CDCV) hypothesis of complex disorders like schizophrenia. In a recent mega GWAS analysis (Ripke et al., 2011), performed by the Psychiatric GWAS Consortium (PGC), the strongest finding for association with schizophrenia was to a variant within the primary transcript of miRNA gene, hsa-miR-137 (miR-137). This miRNA has been previously implicated in regulation of adult neurogenesis (Szulwach et al., 2010), dendritic development, and neuronal maturation (Smrt et al., 2010). In a recent study, using mouse embryonic neural stem cells, miR-137 was shown to control the dynamics between neural stem cell proliferation and differentiation during neural development (Sun et al., 2011). Further, a study integrating GWAS genetic data with brain imaging as a quantitative trait (Potkin et al., 2010) found miR-137 gene targets to be significantly enriched for association with schizophrenia. Additionally, this study also provided evidence for genome-wide significance of association with schizophrenia for four other loci (transcription factor 4 (TCF4), calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C), cub and sushi multiple domains 1(CSMD1) and chromosome 10 open reading frame 26 (C10orf26)) which were also predicted and validated (Kwon et al., 2011) as miR-137 gene targets. MiRNAs mainly function to down-regulate gene expression (Lai, 2002) and genetic studies have found genetic variants within miRNA genes to be associated with schizophrenia as well as expression data performed in postmortem brain tissue have demonstrated dysregulated expression of miRNAs schizophrenic subjects (Burmistrova et al., 2007, Hansen et al., 2007a, Perkins et al., 2007, Beveridge et al., 2010, Kim et al., 2010).

In addition to the PGC study, other GWAS have also provided compelling evidence for association of the ZNF804A gene located at chromosome 2q32.1 with schizophrenia. In the original GWAS an intronic polymorphism, rs1344706, in ZNF804A achieved genome wide significance of association with schizophrenia and bipolar disorder in a combined SZ and BP samples (O'Donovan et al., 2008). This was later corroborated by a meta-analysis of over 21,000 cases and 38,000 controls, which found an odds ratio (OR) of 1.10, P = 2.5 × 10 11 for schizophrenia alone, and OR 1.11, P = 4 × 10 13 for schizophrenia and bipolar disorder combined (Williams et al., 2011).

Given that animal miRNAs bind with imperfect complementarity to their targets and considering the labor intensive approaches to experimentally verify such miRNA targets, much effort has been put toward devising a genome-wide computational search that captures most of the regulatory targets without inflating the rate of false-positive predictions. While, the prediction methods are diverse and all have room for improvement, a general agreement has emerged on three important criteria (Lewis et al., 2003, Brennecke et al., 2005, Bartel, 2009, Huang et al., 2010). First, strong binding of the 5′ seed sequence (nucleotides 2–7) of the mature miRNA to the 3′-UTR sequence of the target gene, second assessing the thermodynamic properties of the miRNA/mRNA duplex by calculating the free-energy (ΔG) of the putative interaction, i.e. a lower ΔG indicating stronger miRNA/mRNA binding and third evolutionary conservation of the miRNA target sequences. Based on these criteria for computational prediction of miRNA/mRNA interactions, many algorithms have been developed and eleven of these well-established algorithms have been compiled into a single database, miRecords (Xiao et al., 2009).

Regardless of the computational algorithms used, the experimental approaches are still the best option to unequivocally establish if a given miRNA interacts with its predicted gene target. Therefore here, using both in silico and cellular based approaches we provide strong evidence that in addition to other schizophrenia implicated genes,TCF4, CACNA1C, CSMD1 and C10orf26, ZNF804A is another target for hsa-mi-137.

Section snippets

In silico analysis

Since the accuracy of in silico predictions is shown to be considerably improved by integrating multiple prediction programs, we used miRecords to predict ZNF804A as a miR-137 gene target. The predicted targets module in miRecords is an integration of 11 established miRNA target prediction programs. Within miRecords database only one prediction algorithm (PITA) (Kertesz et al., 2007) predicted ZNF804A as a gene target for miR-137.

Cell cultures

Be2C (neuroblastoma) cells (ATCC # CRL-2268) were propagated at 37

Computational prediction of ZNF804A as hsa-miR-137 target

ZNF804A was predicted as miR-137 gene target using the miRecords database. Within miRecord, one miR-137 binding site at nucleotide (nt) position 4660 was predicted for ZNF804A (NM_194250.1) by PITA and therefore was used as the major target site for the subsequent experiments.

ZNF804A expression in HEK293 and Be2c cell lines

To assess whether miR-137 down-regulates the native expression of ZNF804A, expression vectors containing the miR-137 hairpin and a SH control were transfected independently into HEK293 and Be2C cell lines. After 24 h, total

Discussion

In the last few years, based on genetic and expression studies, there has been a steady increase in the number of reports demonstrating miRNAs involvement in schizophrenia and bipolar disorders as well as other psychiatric disorders including addiction (Dreyer, 2010, Miller and Wahlestedt, 2010, Xu et al., 2010). In regards to existing genetic studies, the recent mega GWAS study published last year by the PGC group is of special interest as insofar it provides the strongest genetic evidence for

Role of funding source

Funding for this study was provided by SMRI grant (#08R-1959) and Thomas Jeffress & Kate Miller Jeffress Memorial Trust (J-1015) to V.I.V.; the funding bodies had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Funding for A.H.F. was provided by a grant from the Department of Veterans Affairs Merit Review Program.

Contributors

A.H.K., E. K.P. and G. M. performed the experimental work. V.W. performed the in silico analyses. V.I.V. designed the study and wrote the first draft of the manuscript. A.H.F. contributed to the final writing of the manuscript. All authors contributed to and have approved the final manuscript.

Conflict of interest

The authors declare no financial interests or potential conflicts of interest.

Acknowledgments

N/A.

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