It is well known that miRNA levels are tightly controlled and deregulation of miRNAs is a hallmark of impaired brain development and nervous system diseases (Kosik 2006). In addition, miRNAs modulate almost all biological events and signaling cascades (Bartel 2009; Ouellet et al. 2006). miRNA expression profiles of the PVN and supraoptic nuclei of the hypothalamus or various region of the mouse brain including hypothalamus by miRNA microarray methods were previously reported (Lee et al. 2006; Bak et al. 2008). Despite its convenient and reliable use, microarray techniques have limitations such as reliance on known sequences, relatively low sensitivity and specificity, and poor range of quantification. Deep sequencing enables the identification and quantification of small-sized RNAs such as miRNAs, including novel ones (Landgraf et al. 2007; Taft et al. 2009). Using this technique, we analyzed detailed miRNA profiles from adult mouse hypothalamus. The most highly expressed miRNA in the mouse hypothalamus was miR-127, which is known as one of the highest expressed miRNAs in the brain (Lu et al. 2005), though its function there is still unclear. miR-7b (Table S1), abundant in the hypothalamus (Lee et al. 2006; Herzer et al. 2012; Sanek and Young 2012), is up-regulated in response to osmotic stress and inhibits c-fos (known as marker for neuronal activity) by binding 3′ UTR of the c-fos gene (Lee et al. 2006). For this reason, we verified the expression of various hypothalamus miRNAs by qRT-PCR and normalized to miR-7b. Many of the miRNAs that were reported to be influential in brain function were also expressed in relatively high amounts as revealed by our profiling analysis. For example, the miR-9 gene is conserved in vertebrates where it regulates the midbrain–hindbrain boundary formation and, with miR-124, plays a role in neuronal differentiation (Leucht et al. 2008). The expression of miR-874 is dramatically reduced in glioblastoma tissues, compared with normal brain tissues. miR-124, another miRNA that is down-regulated in glioblastoma stem cells, also exhibits reduced expression in glioblastoma tissues by deep sequencing (Cheng et al. 2012). Among the highly expressed miRNAs, we used bioinformatic tools to find possible miRNAs that could regulate Oxt expression. As originally described, targeting to inhibit mRNAs by miRNAs is accomplished by base pairing between the 3′ UTRs of the target genes and miRNAs (Robins et al. 2005). However, recently, many studies have found other sites of mRNA interaction including the coding sequence (Qin et al. 2010; Tay et al. 2008; Forman et al. 2008) and 5′ UTR (Lee et al. 2009b). Interestingly, the miR-24 target site in Oxt spans the boundary between the coding sequence and 3′ UTR (Fig. 3a) and miR-24 was the only miRNA that showed significant binding activity by luciferase assay among the miRNAs predicted from various programs (Fig. 3b). Interestingly, one of the Oxt-targeting candidate miRNAs, miR-540 showed up-regulated luciferase activity rather than down-regulation (Fig. 3b). This can be explained by RNA activation (RNAa) (Pushparaj et al. 2008; Lee 2013; Huang et al. 2010), i.e., miR-540 interacted with promoter of the Renilla luciferase gene and thereby, resulted the elevated luciferase activity. However, whether miR-540 can also interact with Oxt needs investigation.
One study has shown that miR-24 can regulate apoptosis by targeting the coding sequence of the FAF1 gene (Qin et al. 2010). Targeting other than the 3′ UTR could be one mechanism by which miRNAs could regulate genes that have short 3′ UTRs, like Oxt. Furthermore, miR-24 reduced both transcript and peptide levels of Oxt (Fig. 4a and b), suggesting its non-canonical regulation (Lee 2013). We also tried to find an miRNA candidate from the profiled sequences that may be able to target Oxtr and Avp, but failed to verify it at both the transcriptional and translational level; although miR-873 could reduce luciferase activity by binding to the Oxtr 3′ UTR (data not shown) and needs further investigation of any relevance between Oxtr and miR-873.
Deep sequencing profile analysis of annotated clones resulted in certain novel miRNA candidates although the read counts were extremely low. However, we cannot exclude the possibility that those novel miRNA candidates could be induced by neuronal stimuli and therefore functionally activated. The structure and biological significance of these candidates need to be elucidated in future studies.
It is well known that steroid hormones can regulate Oxt and Oxtr (Lee et al. 2009a; Choleris et al. 2003). Oxt and Oxtr promoters of many species harbor binding sites for the estrogen receptor (estrogen-response elements) and are stimulated by estrogen and thyroid hormones (Mohr and Schmitz 1991; Richard and Zingg 1990; Bale and Dorsa 1995; Lee et al. 2009a). The promoter region of the Oxt gene is important for its cell-specific expression, but the precise cis-domains responsible for gene expression are unclear (Gainer 2012). However, no regulatory mechanism for Oxt expression has been fully characterized.
We propose here that miR-24, an abundant miRNA in hypothalamus, could be a possible regulator of Oxt. Our data showed that miR-24 could target the boundary region between the coding sequence and 3′ UTR and inhibit Oxt production. Deep sequencing analysis can be used to study miRNA gene profiling from selected brain regions and can help collect useful data to predict region-specific miRNAs that regulate neural gene expression.