Effect of (S)-3,5-DHPG on microRNA expression in mouse brain

https://doi.org/10.1016/j.expneurol.2012.01.018Get rights and content

Abstract

MicroRNAs are small non-coding RNAs that regulate post-transcriptional gene expression. In the short time since the discovery of microRNAs, the literature has burgeoned with studies focused on the biosynthesis of microRNAs, target prediction and binding, and mechanisms of translational repression by microRNAs. Given the prominent role of microRNAs in all areas of cell biology, it is not surprising that microRNAs are also linked to human diseases, including those of the nervous system. One of the least-studied areas of microRNA research is how their expression is regulated outside of development and cancer. Thus, we examined a role for regulation of microRNAs by neurotransmitter receptor activation in mouse brain. We focused on the group I metabotropic glutamate receptors by using intracerebroventricular injection of the selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) in mouse brain. We then examined the expression of microRNAs in the cerebral cortex by Ambion and Invitrogen microarrays, and the expression of mature microRNA sequences by SABiosciences qPCR arrays, at 4, 8 and 24 h after DHPG injection. These studies revealed that the largest number of significantly regulated microRNAs was detected 8 h after DHPG injection in the microarrays and qPCR arrays. We then used RNA blots to quantify microRNA expression, and in situ hybridization to examine cellular distribution of the microRNAs regulated by DHPG. Bioinformatic analysis of the microRNAs regulated 8 h after DHPG in all three arrays revealed KEGG pathways that are known to correlate with group I mGluR effects, as well as recently described and novel pathways. These studies are the first to show that DHGP regulates the expression of microRNAs in mouse cerebral cortex, and support the hypothesis that group I mGluRs may regulate microRNA expression in mouse brain.

Introduction

MicroRNAs are a class of small, non-coding RNAs that function to regulate post-transcriptional gene expression. MicroRNAs bind to complementary sequences in the 3′-untranslated region (3′UTR) of target messenger RNA (mRNA) transcripts, resulting in translational repression and/or accelerated mRNA destabilization (Guo et al., 2010). The miRBase Sequence Database Release 17 contains 16,772 entries representing hairpin precursor miRNAs, expressing 19,724 mature miRNA products, in 153 species (http://www.mirbase.org/) (Griffiths-Jones et al., 2008). Approximately one-half of microRNA genes are contained within introns of protein-coding transcripts, and they can be differentially processed from the sense and antisense strands of the same hairpin RNA or transcripts from the same locus (Amaral et al., 2008). MicroRNAs are transcribed into primary microRNA transcripts, cleaved to 60–70 nucleotides in the nucleus, and the resulting precursor microRNAs are actively transported to the cytoplasm. There they are cleaved by endonucleases such as Dicer to produce mature microRNAs which bind to ribonucleoproteins to form RNA-induced silencing complexes (RISCs). MicroRNAs in RISCs target ~ 60% of mammalian genes (Friedman et al., 2009).

There has been intense focus on determining the mechanisms of microRNA-regulated post-transcriptional gene expression, and their roles in development, brain function, and brain disorders (Potkin et al., 2010, Provost, 2010, Rachidi and Lopes, 2010, Satoh, 2010, Saugstad, 2010, Sonntag, 2010, Yelamanchili and Fox, 2010). However, less attention has been focused on the mechanisms which regulate microRNA expression. A recent study revealed that long-term potentiation induced by high-frequency stimulation of the medial perforant pathway and activation of mGluRs and NMDARs resulted in differential regulation of primary and mature microRNAs (Wibrand et al., 2010). Thus, we examined the effect of intracerebroventricular (ICV) injection of the group I mGluR-selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), on microRNA expression in mouse brain. We then used Ambion and Invitrogen microRNA microarrays, as well as the SABiosciences quantitative PCR (qPCR) arrays to profile microRNA expression in total RNA isolated from the cerebral cortex. Further, we used in situ hybridization to examine the anatomical distribution, and RNA blot analysis to quantify expression of, select microRNAs in DHPG-treated mouse brain. In addition, we used KEGG analysis to examine pathways that are potentially regulated by the microRNAs significantly altered by DHPG. These studies are the first to show that DHPG regulates the expression of microRNAs in mouse cerebral cortex, and support a potential role for group I mGluRs in the regulation of microRNA expression in mouse brain.

Section snippets

Materials

DHPG was purchased from Tocris Bioscience (Ellisville, MO). The mirVana™ miRNA Isolation Kits were purchased from Applied Biosystems/Ambion (Austin, TX). The Mouse MicroRNA Genome V2.0 PCR Arrays (MAM-200C) were purchased from SABiosciences (Frederick, MD). The 5′ Digoxigenin (DIG)-labeled Locked Nucleic Acid (LNA) detection probes (scrambled: #99004, hsa-miR-132: #38031, mmu-miR-463: #39591, mmu-miR-709: #39324, has-miR-200c: #38536, hsa-miR-19a: #18091; U6: #99002) were purchased from Exiqon

Regulation of MicroRNAs by DHPG detected by Ambion and Invitrogen microarrays

We used the Ambion/Invitrogen (AI) microarray to analyze the relative expression of all microRNA forms expressed in naïve, saline-injected (8 h), and DHPG-injected (4, 8 and 24 h) mice. The AI arrays contain the complement to mature microRNA sequence, thus they will detect mature sequences regardless of whether they are part of a mature, precursor, or primary microRNA. Of the 421 microRNA probes on the AI microarray set, a total of 226 were detected in our experiments. Analysis of the microarray

Discussion

Little is known about the response of microRNAs as a result of specific receptor signaling. Microarray studies have shown regulated expression of hippocampal microRNAs in response to the induction of mGluR-dependent LTD (Park and Tang, 2008) or LTP (Wibrand et al., 2010). Thus, we examined the direct effects of the group I mGluR-specific agonist, DHPG on microRNA expression by ICV injection in adult mouse brain, a well-established model for examining in vivo responses. We profiled microRNA

Acknowledgments

The authors acknowledge the generous support of NIH grants NS050221, NS054220, and NS064270 (JAS).

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