Downregulation of selective microRNAs in trigeminal ganglion neurons following inflammatory muscle pain
© Bai et al; licensee BioMed Central Ltd. 2007
Received: 04 May 2007
Accepted: 08 June 2007
Published: 08 June 2007
Active regulation of gene expression in the nervous system plays an important role in the development and/or maintenance of inflammatory pain. MicroRNA (miRNA) negatively regulates gene expression via posttranscriptional or transcriptional inhibition of specific genes. To explore the possible involvement of miRNA in gene regulation during inflammatory pain, we injected complete Freund's adjuvant (CFA) unilaterally into the rat masseter muscle and quantified changes in neuron-specific mature miRNAs in the trigeminal ganglion (TG). Real-time reverse-transcription polymerase chain reaction revealed significant, but differential, downregulation of mature miR-10a, -29a, -98, -99a, -124a, -134, and -183 in the ipsilateral mandibular division (V3) of the TG within 4 hr after CFA. In contrast, levels of tested miRNAs did not change significantly in the contralateral V3 or the ipsilateral ophthalmic and maxillary divisions of the TG from inflamed rats, nor in the ipsilateral V3 of saline-injected animals. The downregulated miRNAs recovered differentially to a level equal to or higher than that in naive animals. Full recovery time varied with miRNA species but was at least 4 days. Expression and downregulation of some miRNAs were further confirmed by in situ hybridization of TG neurons that innervate the inflamed muscle. Although neurons of all sizes expressed these miRNAs, their signals varied between neurons. Our results indicate that miRNA species specific to neurons are quickly regulated following inflammatory muscle pain.
Inflammation associated with some pathologies may develop allodynia or hyperalgesia defined as an over-reaction to non-noxious or noxious stimuli, respectively [1, 2]. Gene expression is an important molecular mechanism underlying inflammatory pain since the measured steady-state levels of mRNA and/or protein in pain/nociceptive pathway in animal models are actively altered during the development and maintenance of pain [2–6]. Our understanding of how individual genes are selectively regulated during inflammatory pain is limited mostly to the regulation of transcriptional control . MicroRNA (miRNA) represents a group of small noncoding RNAs in 18~23 nucleotide sequences. These evolutionarily conserved molecules mainly interfere with gene expression at posttranscriptional levels and moderately promote RNA degradation by acting on specific sequences in the 3' untranslated region of target mRNA, while some of them inhibit gene transcription by participating in chromatin remodeling [7–9]. While many miRNAs have been detected in the nervous system [10–12], their functional significance has been restricted mostly to events involving nervous system development [10, 13–18]. Although miRNAs are present in mature neurons, their functionality and regulation remain largely unexplored.
To explore the mechanism(s) underlying the gene alteration during inflammatory pain and to investigate the function of miRNA in the adult nervous system, we quantified several neuronal miRNAs in a model of inflammatory muscle pain. We injected CFA (150 μl, oil:saline = 1:1, Sigma, St. Louis, MO) unilaterally into the masseter muscle of male rats (Sprague-Dawley, ~250 gr, Harlan, Indianapolis, IN). This injection produced significant mechanical allodynia while intramuscular injection of saline did not [4, 6]. After the development of allodynia, we dissected the ophthalmic and maxillary divisions (V1/2) and V3 from individual TGs. Tissues were combined from two animals and cellular RNA was extracted for miRNA quantification . This design is based on the hypothesis that sensory neurons are a critical component in pain/nociception pathway  and sensory neurons innervating mandibular muscle have their perikarya located in V3 . To quantify miRNA, we employed a newly developed TaqMan real-time reverse-transcription polymerase chain reaction (RT-PCR) assay (ABI, Foster City, CA). This technology allows us to specifically measure selective mature miRNAs from nanogram amounts of cellular RNA, thus making it possible to study small tissues such as dissected TG . In the present study we used the following criteria to limit miRNA number from more than 400 identified molecules : First, miRNAs expressed in TG were included. Seven miRNAs were reported previously from TG . Second, those involved in neuronal plasticity , one of cellular mechanisms underlying inflammatory pain , were chosen. Third, one member per miRNA family was examined [11, 12, 23]. Fourth, the amount of extracted RNA and the availability of relevant TaqMan miRNA assays limited the number of miRNA tested. Last, they are conserved among human and rodents. In preliminary studies, we examined ten miRNAs from a pool of RNA extracted from TG V3 (n = 16). We were able to detect miR-10a, -29a, -98, -99a, -124a, -134, and -183, but not miR-122, miR-143, and miR-153 even after 50 cycles of PCR, although they were previously reported from TG via Northern analysis . In parallel experiments, all assays produced robust signal from brain RNA (data not shown).
The LNA probe is virtually antisense to the mature miRNA sequence that is present in both pre- and mature miRNA in the cytosol . Therefore, the signal obtained from in situ hybridization represents both types of molecules of a specific miRNA. The results of the TaqMan assay and in situ hybridization suggest that the downregulation occurred either at both pre- and mature miRNA levels or only at the mature miRNA level if the latter is the major form in the TG.
The present study for the first time demonstrates miRNA expression in the peripheral nervous system at the mature miRNA level and with single cell resolution. Most importantly, we observed that several miRNA molecules, likely in the mature form, are regulated by an inflammatory irritant and their changes are correlated with the development of allodynia. Although the detailed mechanism underlying this regulation remains unknown at this stage, the RNA polymerase II (Pol II) is found to govern the transcription of the most miRNA genes , and inflammation is known to induce rapid expression or modification of several transcription factors such as c-fos and CREB in neurons [2, 3, 6]. These factors may negatively regulate Pol II activity in neurons under certain conditions.
Discovery of miRNA downregulation provides a novel view of the mechanism(s) underlying inflammatory pain. Downregulation of miRNA releases the translation inhibition of target mRNAs, thus yielding more proteins that may be relevant to the development and/or maintenance of inflammatory pain. However, these initial studies only demonstrated downregulation of a few selected miRNAs in TG sensory neurons during the time when allodynia occurred . Whether this miRNA downregulation is mechanistically involved in inflammatory pain cannot be addressed by the present study. How miRNA participates in inflammatory pain relies, at least in part, on the elucidation of their target mRNAs and/or on the impact of manipulated levels of specific miRNA on nociception. The former is a complex question. Even though several programs have been developed to predict the potential targets for a given miRNA [23, 25–27], systematic studies are needed to thoroughly address this question.
This work is supported by NIH grants NS38077, DE15386 and DE016795.
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