MicroRNAs are little non-coding RNAs that regulate post-transcriptional gene expression. cortex,
MicroRNAs are little non-coding RNAs that regulate post-transcriptional gene expression. 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 (3UTR) 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 Darapladib supplier 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 60C70 nucleotides in the nucleus, and the resulting precursor microRNAs are actively transported to the cytoplasm. There they are cleaved by endonucleases such as 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 functions 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 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, (hybridization to examine the anatomical distribution, and RNA blot analysis to Darapladib supplier quantify expression of, select microRNAs in DHPG-treated mouse brain. In addition, we used KEGG analysis to examine pathways that are controlled with the microRNAs significantly altered by DHPG potentially. These studies will be the first showing that DHPG regulates the appearance of microRNAs in mouse cerebral cortex, and support a potential function for group I in the regulation of microRNA expression in mouse human brain mGluRs. MATERIALS AND Strategies Components DHPG was bought from Tocris Bioscience (Ellisville, MO). The hybridizations, and RNA blots to investigate the appearance of microRNAs in mouse human brain after ICV shot of DHPG. A listing of the experimental style for Darapladib supplier everyone scholarly research is listed in Desk 1. Sample groupings included five band of mice: (i) na?ve control, (ii) saline injected – 8 hours, (iii) DHPG injected – 4 hours, (iv) DHPG injected – 8 hours, and (v) DHPG injected – a day, ahead of sacrifice. Table 1 Experimental Design Intracerebroventricular Injection of DHPG Adult male C57BL/6J mice (25C30 g; Charles River Laboratories, Wilmington, MA) were housed and cared for in the Animal Care Facility of Legacy Research, and all procedures were performed in an AAALAC accredited facility in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols and principles layed out in the National Institutes of Health hybridization Darapladib supplier studies, the mice were deeply anesthetized using isoflurane and transcardially perfused with new filter-sterilized 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO) in 1X Tris-buffered saline (TBS, pH 7.0). The whole brain was isolated and incubated at 4C overnight in new 4% PFA in 1X TBS, cryoprotected by incubation in sterile 30% sucrose in 1X TBS for 72 hours, then the tissues were frozen on dry ice and stored at ?80C until further use. RNA Isolation and Quality Assessment Total RNA Mouse monoclonal to MYL3 was isolated from the right and left mouse cortex using the mirVana? kit (Ambion, Austin, TX) according to the manufacturers instructions and as previously explained (Lusardi, et al., 2010)..