微小核糖核酸是一種非編碼的核糖核酸，可在轉譯階段調節基因表現，而且已知在神經系統發育的過程佔重要的地位。最近的一些研究則是利用微陣列的分析，來探討微小核糖核酸在疼痛產生路徑裡的表現。但是，被微小核糖核酸調控的基因，傳統上可以利用軟體來預測，但是往往有相當程度的偽陽性。為了提高預測的正確性和增加目標基因的數量，本次的實驗是在大鼠的腳掌注射Complete Freund’s adjuvant (CFA)誘發發炎性疼痛後的第五天和第十四天，取大鼠脊髓背根做微小核糖核酸(microRNA)和傳訊核糖核酸(mRNA)的微陣列(microarray)分析，然後將其結果進行統合性分析。以出現1.5倍以上變化的表現量為篩選標準，在注射CFA後第五天 (CFA 5d) 這組，有5個微小核糖核酸和1096個傳訊核糖核酸納入後續的分析；而注射CFA後在第十四天 (CFA 14d) 這組，則有16個微小核糖核酸和647個傳訊核糖核酸。進行統合性分析後，在CFA 5d這組，發現有54個傳訊核糖核酸可能被3個微小核糖核酸所調控，而在CFA 14d這組，則有75個傳訊核糖核酸可能被6個微小核糖核酸調控。在CFA 5d這組所顯示的微小核糖核酸與傳訊核糖核酸的交互作用網路中，發現miR-124, miR-149和miR-3584這三個傳訊核糖核酸主要是調控IL6R, ADAM19, LAMC1和CERS2這些基因。而在CFA 14d這組，miR-124, miR-29, miR-34, miR-30和miR-338則是調控TIMP2, CREB5和EFNB1這些基因。在CFA 5d這組，IL6R (細胞介素6接受器) 被預測是miR-124-3p的目標基因；IL6R被認為和疼痛的發生有關，因為抑制IL6R可以減少脊髓損傷後產生的觸覺痛 (allodynia)和痛覺敏感 (hyperalgesia)。因此本實驗進而探討miR-124對IL6R 的調控，結果發現miR-124可以減緩發炎性疼痛，同時造成IL6R的表現量降低。因此，這些特定的微小核糖核酸和他們的目標基因(傳訊核糖核酸)應該可以提供未來治療發炎性疼痛時一個很好的方向。

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression involved in fundamental cell processes. Recent studies using microarray-based approaches have demonstrated that microRNAs (miRNAs) are involved in pain processing pathways. However, a significant proportion of computational predictions of miRNA targets are false-positive interactions. To increase the chance of identifying biologically relevant targets, we performed an integrated analysis of both miRNA and mRNA expression profiles in the rat spinal cord during complete Freund's adjuvant (CFA)-induced inflammatory pain. We generated miRNA and mRNA arrays from the same corresponding samples on Days 5 and 14 after CFA injection. Five miRNAs and 1096 mRNAs in the CFA 5d group and 16 miRNAs and 647 mRNAs in the CFA 14d group were differentially expressed based on a filter of at least a 1.5-fold change in either direction. An integrated analysis revealed 54 mRNA targets with an inverse correlation to the expression patterns of 3 miRNAs in the CFA 5d group. Seventy-five targets were inversely correlated to 6 miRNAs in the CFA 14d group. The miRNA-mRNA interaction networks revealed significant changes in miR-124, miR-149, miR-3584 and their target genes, IL6R, ADAM19, LAMC1 and CERS2, in the CFA 5d group. In the CFA 14d group, significant changes were noted in miR-124, miR-29, miR-34, miR-30, miR-338 and their target genes, TIMP2, CREB5 and EFNB1. IL6R may play a role in remodeling inflammation-driven pain neuro-circuitry following SCI (spinal cord injury). Besides, continuous inhibition of IL6 signaling using an anti-mouse IL6R antibody between the early and sub-acute phases following SCI reduced damaging inflammatory activity and suppressed hyperalgesia and allodynia in mice. We also investigated an interaction pair, miR-124-3p and IL6R, and the results showed that miR-124-3p could attenuate inflammatory pain and decrease IL6R expression in the spinal cord. These specific miRNAs and their target genes provide possible avenues for the diagnosis and treatment of inflammatory pain.

論文審定書 i
論文公開授權書 ii
誌謝 iii
中文摘要 iv
Abstract v
Table of contents 目錄 vii
List of figures 圖次 ix
List of tables 表次 x
Chapter 1. Background 1
1.1 Introduction 1
1.2 The mechanism of miRNA biogenesis 2
1.3 The mechanism of chronic pain 3
1.4 The regulatory role of miRNAs in pain processing pathway 5
1.5 miRNAs in inflammatory pain 6
1.6 Conclusions 8
Chapter 2. Research objectives 9
2.1 Introduction 9
2.2 Data integration in functional analysis of microRNAs 10
2.3 Research objectives and specific aims 11
2.3.1 Aim 1: Investigating expression profiles of miRNAs and mRNAs in the same corresponding samples of CFA-induced inflammatory rat model 11
2.3.2 Aim 2: Integration analysis of differential expressed miRNAs and mRNA 11
2.3.3 Aim 3: Verifying the predicted miRNA-mRNA interactive network 11
2.4 Experimental design 11
Chapter 3. Materials and methods 12
3.1 Animals 12
3.2 Drugs and administration 12
3.3 CFA-induced inflammation and experimental groups 13
3.3.1 CFA-induced inflammation 13
3.3.2 Experimental groups 13
3.4 Behavioral tests 14
3.4.1 The von Frey test. 14
3.4.2 The plantar test. 14
3.5 Total RNA isolation 15
3.6 miRNA microarray analysis 15
3.7 Microarray profiling of gene expression 16
3.8 Enriched biological function analysis 17
3.9 Quantitative real-time PCR (qPCR) assay 18
3.10 Western blot analysis 18
3.11 Statistics 19
Chapter 4. Results 20
4.1 Behavioral change after CFA injection 20
4.2 Differentially expressed miRNAs and mRNAs in SDHs 20
4.3 Integrated analysis of differentially expressed miRNAs and mRNAs 21
4.4 Validation of selected miRNAs from the integrated analysis results 22
4.5 Biologically enriched functions of inflammatory pain regulated by mRNAs and miRNAs 22
4.6 miRNA and mRNA interaction networks in inflammatory pain 24
4.7 Validation of miR-124-3p and IL6R interaction 26
4.8 Time course of the analgesic effect of miR-124-3p 27
Chapter 5. Discussion 28
5.1 The advantage of an integrated analysis 28
5.2 The discordance between microarray and qPCR results 29
5.3 The interaction pair of miR-124-3p and IL6R in early phase of inflammatory pain 30
5.4 The other interesting pairs in early phase of inflammatory pain 31
5.5 The interaction networks in late phase of inflammatory pain 32
5.6 Limitations of this study 33
5.7 Conclusions 33
References 34
Figures and Legands 47
Tables 64

Ach, R.A., Wang, H. & Curry, B. (2008) Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods. BMC Biotechnol, 8, 69.

Alexiou, P., Maragkakis, M., Papadopoulos, G.L., Reczko, M. & Hatzigeorgiou, A.G. (2009) Lost in translation: an assessment and perspective for computational microRNA target identification. Bioinformatics, 25, 3049-3055.

Ambros, V. (2004) The functions of animal microRNAs. Nature, 431, 350-355.

Anderson, L.E. & Seybold, V.S. (2000) Phosphorylated cAMP response element binding protein increases in neurokinin-1 receptor-immunoreactive neurons in rat spinal cord in response to formalin-induced nociception. Neurosci Lett, 283, 29-32.

Artmann, S., Jung, K., Bleckmann, A. & Beissbarth, T. (2012) Detection of simultaneous group effects in microRNA expression and related target gene sets. PLoS One, 7, e38365.

Bai, G., Ambalavanar, R., Wei, D. & Dessem, D. (2007) Downregulation of selective microRNAs in trigeminal ganglion neurons following inflammatory muscle pain. Mol Pain, 3, 15.

Bali, K., Selvaraj, D., Satagopam, V.P., Lu, J., Schneider, R. & Kuner, R. (2013) Genome wide identification and functional analyses of microRNA signatures associated with cancer pain. EMBO molecular medicine, 5, 1740-1758.

Bali, K.K., Hackenberg, M., Lubin, A., Kuner, R. & Devor, M. (2014) Sources of individual variability: miRNAs that predispose to neuropathic pain identified using genome-wide sequencing. Mol. Pain.

Barbato, C., Arisi, I., Frizzo, M.E., Brandi, R., Da Sacco, L. & Masotti, A. (2009) Computational challenges in miRNA target predictions: to be or not to be a true target? J Biomed Biotechnol, 2009, 803069.

Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116, 281-297.

Bartel, D.P. (2009) MicroRNAs: target recognition and regulatory functions. Cell, 136, 215-233.

Becker, I., Wang-Eckhardt, L., Yaghootfam, A., Gieselmann, V. & Eckhardt, M. (2008) Differential expression of (dihydro)ceramide synthases in mouse brain: oligodendrocyte-specific expression of CerS2/Lass2. Histochem Cell Biol, 129, 233-241.

Beggs, S., Liu, X.J., Kwan, C. & Salter, M.W. (2010) Peripheral nerve injury and TRPV1-expressing primary afferent C-fibers cause opening of the blood-brain barrier. Mol Pain, 6, 74.

Bentwich, I. (2005) Prediction and validation of microRNAs and their targets. FEBS Lett, 579, 5904-5910.

Betel, D., Wilson, M., Gabow, A., Marks, D.S. & Sander, C. (2008) The microRNA.org resource: targets and expression. Nucleic Acids Res, 36, D149-153.

Bhalala, O.G., Srikanth, M. & Kessler, J.A. (2013) The emerging roles of microRNAs in CNS injuries. Nat Rev Neurol, 9, 328-339.

Bian, S. & Sun, T. (2011) Functions of noncoding RNAs in neural development and neurological diseases. Mol Neurobiol, 44, 359-373.

Bingham, B., Ajit, S.K., Blake, D.R. & Samad, T.A. (2009) The molecular basis of pain and its clinical implications in rheumatology. Nat Clin Pract Rheumatol, 5, 28-37.

Bohnsack, M.T., Czaplinski, K. & Gorlich, D. (2004) Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA, 10, 185-191.

Bossel Ben-Moshe, N., Avraham, R., Kedmi, M., Zeisel, A., Yitzhaky, A., Yarden, Y. & Domany, E. (2012) Context-specific microRNA analysis: identification of functional microRNAs and their mRNA targets. Nucleic Acids Res, 40, 10614-10627.

Brandenburger, T., Castoldi, M., Brendel, M., Grievink, H., Schlosser, L., Werdehausen, R., Bauer, I. & Hermanns, H. (2012) Expression of spinal cord microRNAs in a rat model of chronic neuropathic pain. Neurosci Lett, 506, 281-286.

Calvo, M. & Bennett, D.L. (2012) The mechanisms of microgliosis and pain following peripheral nerve injury. Exp Neurol, 234, 271-282.

Calvo, M., Dawes, J.M. & Bennett, D.L. (2012) The role of the immune system in the generation of neuropathic pain. Lancet Neurol, 11, 629-642.

Cao, L. & DeLeo, J.A. (2008) CNS-infiltrating CD4+ T lymphocytes contribute to murine spinal nerve transection-induced neuropathic pain. Eur J Immunol, 38, 448-458.

Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M. & Yaksh, T.L. (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods, 53, 55-63.

Chou, C.H., Chang, N.W., Shrestha, S., Hsu, S.D., Lin, Y.L., Lee, W.H., Yang, C.D., Hong, H.C., Wei, T.Y., Tu, S.J., Tsai, T.R., Ho, S.Y., Jian, T.Y., Wu, H.Y., Chen, P.R., Lin, N.C., Huang, H.T., Yang, T.L., Pai, C.Y., Tai, C.S., Chen, W.L., Huang, C.Y., Liu, C.C., Weng, S.L., Liao, K.W., Hsu, W.L. & Huang, H.D. (2016) miRTarBase 2016: updates to the experimentally validated miRNA-target interactions database. Nucleic Acids Res, 44, D239-247.

Clark, A.K., Old, E.A. & Malcangio, M. (2013) Neuropathic pain and cytokines: current perspectives. J Pain Res, 6, 803-814.

Dixon, W.J. (1980) Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol, 20, 441-462.

Echeverry, S., Shi, X.Q., Rivest, S. & Zhang, J. (2011) Peripheral nerve injury alters blood-spinal cord barrier functional and molecular integrity through a selective inflammatory pathway. J Neurosci, 31, 10819-10828.

Ellis, A. & Bennett, D.L. (2013) Neuroinflammation and the generation of neuropathic pain. Br J Anaesth, 111, 26-37.

Engelmann, J.C. & Spang, R. (2012) A least angle regression model for the prediction of canonical and non-canonical miRNA-mRNA interactions. PLoS One, 7, e40634.

Fan, W., Huang, F., Zhu, X., Dong, W., Gao, Z., Li, D. & He, H. (2010) Involvement of microglial activation in the brainstem in experimental dental injury and inflammation. Arch Oral Biol, 55, 706-711.

Fang, Y., Rowe, T., Leon, A.J., Banner, D., Danesh, A., Xu, L., Ran, L., Bosinger, S.E., Guan, Y., Chen, H., Cameron, C.C., Cameron, M.J. & Kelvin, D.J. (2010) Molecular characterization of in vivo adjuvant activity in ferrets vaccinated against influenza virus. J Virol, 84, 8369-8388.

Friedman, R.C., Farh, K.K., Burge, C.B. & Bartel, D.P. (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res, 19, 92-105.

Ghildiyal, M. & Zamore, P.D. (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet, 10, 94-108.

Guo, H., Ingolia, N.T., Weissman, J.S. & Bartel, D.P. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature, 466, 835-840.

Guo, W., Wang, H., Watanabe, M., Shimizu, K., Zou, S., LaGraize, S.C., Wei, F., Dubner, R. & Ren, K. (2007) Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain. J Neurosci, 27, 6006-6018.

Ha, M. & Kim, V.N. (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol, 15, 509-524.

Hammond, S.M., Bernstein, E., Beach, D. & Hannon, G.J. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293-296.

Hargreaves, K., Dubner, R., Brown, F., Flores, C. & Joris, J. (1988) A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain, 32, 77-88.

Huang, B., Zhao, X., Zheng, L.B., Zhang, L., Ni, B. & Wang, Y.W. (2011) Different expression of tissue inhibitor of metalloproteinase family members in rat dorsal root ganglia and their changes after peripheral nerve injury. Neuroscience, 193, 421428.

Hutcheson, D.A. & Vetter, M.L. (2002) Transgenic approaches to retinal development and function in Xenopus laevis. Methods, 28, 402-410.

Hutvagner, G. & Zamore, P.D. (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science, 297, 2056-2060.

Hylden, J.L. & Wilcox, G.L. (1980) Intrathecal morphine in mice: a new technique. Eur J Pharmacol, 67, 313-316.

Jensen, T.S. & Finnerup, N.B. (2014) Allodynia and hyperalgesia in neuropathic pain: clinical manifestations and mechanisms. Lancet Neurol, 13, 924-935.

Ji, R.R., Kohno, T., Moore, K.A. & Woolf, C.J. (2003) Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci, 26, 696-705.

Kawasaki, Y., Xu, Z.-Z., Wang, X., Park, J., Zhuang, Z.-Y., Tan, P.-H., Gao, Y.-J., Roy, K., Corfas, G. & Lo, E.H. (2008) Distinct roles of matrix metalloproteases in the early-and late-phase development of neuropathic pain. Nature medicine, 14, 331-336.

Kiyomoto, M., Shinoda, M., Okada-Ogawa, A., Noma, N., Shibuta, K., Tsuboi, Y., Sessle, B.J., Imamura, Y. & Iwata, K. (2013) Fractalkine signaling in microglia contributes to ectopic orofacial pain following trapezius muscle inflammation. J Neurosci, 33, 7667-7680.

Kobayashi, Y., Kiguchi, N., Maeda, T., Ozaki, M. & Kishioka, S. (2012) The critical role of spinal ceramide in the development of partial sciatic nerve ligation-induced neuropathic pain in mice. Biochem Biophys Res Commun, 421, 318-322.

Krek, A., Grun, D., Poy, M.N., Wolf, R., Rosenberg, L., Epstein, E.J., MacMenamin, P., da Piedade, I., Gunsalus, K.C., Stoffel, M. & Rajewsky, N. (2005) Combinatorial microRNA target predictions. Nat Genet, 37, 495-500.

Krol, J., Loedige, I. & Filipowicz, W. (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet, 11, 597-610.

Kusuda, R., Cadetti, F., Ravanelli, M.I., Sousa, T.A., Zanon, S., De Lucca, F.L. & Lucas, G. (2011) Differential expression of microRNAs in mouse pain models. Mol Pain, 7, 17.

Kynast, K.L., Russe, O.Q., Moser, C.V., Geisslinger, G. & Niederberger, E. (2013) Modulation of central nervous system-specific microRNA-124a alters the inflammatory response in the formalin test in mice. Pain, 154, 368-376.

Lander, A.D., Fujii, D.K. & Reichardt, L.F. (1985) Laminin is associated with the "neurite outgrowth-promoting factors" found in conditioned media. Proc Natl Acad Sci U S A, 82, 2183-2187.

Laviad, E.L., Albee, L., Pankova-Kholmyansky, I., Epstein, S., Park, H., Merrill, A.H., Jr. & Futerman, A.H. (2008) Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate. J Biol Chem, 283, 5677-5684.

Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S. & Kim, V.N. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature, 425, 415-419.

Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, C.B. (2003) Prediction of mammalian microRNA targets. Cell, 115, 787-798.

Li, X., Gibson, G., Kim, J.S., Kroin, J., Xu, S., van Wijnen, A.J. & Im, H.J. (2011) MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis. Gene, 480, 34-41.

Li, X., Kroin, J.S., Kc, R., Gibson, G., Chen, D., Corbett, G.T., Pahan, K., Fayyaz, S., Kim, J.S., van Wijnen, A.J., Suh, J., Kim, S.G. & Im, H.J. (2013) Altered spinal microRNA-146a and the microRNA-183 cluster contribute to osteoarthritic pain in knee joints. J Bone Miner Res, 28, 2512-2522.

Liu, N.K., Wang, X.F., Lu, Q.B. & Xu, X.M. (2009) Altered microRNA expression following traumatic spinal cord injury. Exp Neurol, 219, 424-429.

Ma, W. & Quirion, R. (2001) Increased phosphorylation of cyclic AMP response element-binding protein (CREB) in the superficial dorsal horn neurons following partial sciatic nerve ligation. Pain, 93, 295-301.

Marchand, F., Perretti, M. & McMahon, S.B. (2005) Role of the immune system in chronic pain. Nat Rev Neurosci, 6, 521-532.

Masae, A., Yuuki, G., Masashi, I., Tanaka, S., Tadashi, O. & Atsuhiro, S. (2013) The miRNA and mRNA Changes in Rat Hippocampi after Chronic Constriction Injury. Pain Medicine, 14, 720-729.

Mattick, J.S. & Makunin, I.V. (2006) Non-coding RNA. Hum Mol Genet, 15 Spec No 1, R17-29.

Medzhitov, R. (2008) Origin and physiological roles of inflammation. Nature, 454, 428-435.

Messersmith, D.J., Kim, D.J. & Iadarola, M.J. (1998) Transcription factor regulation of prodynorphin gene expression following rat hindpaw inflammation. Brain Res Mol Brain Res, 53, 260-269.

Miletic, G., Pankratz, M.T. & Miletic, V. (2002) Increases in the phosphorylation of cyclic AMP response element binding protein (CREB) and decreases in the content of calcineurin accompany thermal hyperalgesia following chronic constriction injury in rats. Pain, 99, 493-500.

Mor, E., Cabilly, Y., Goldshmit, Y., Zalts, H., Modai, S., Edry, L., Elroy-Stein, O. & Shomron, N. (2011) Species-specific microRNA roles elucidated following astrocyte activation. Nucleic Acids Res, 39, 3710-3723.

Morey, J.S., Ryan, J.C. & Van Dolah, F.M. (2006) Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online, 8, 175-193.

Murakami, T., Kanchiku, T., Suzuki, H., Imajo, Y., Yoshida, Y., Nomura, H., Cui, D., Ishikawa, T., Ikeda, E. & Taguchi, T. (2013) Anti-interleukin-6 receptor antibody reduces neuropathic pain following spinal cord injury in mice. Exp Ther Med, 6, 1194-1198.

Nakazawa, T., Watabe, A.M., Tezuka, T., Yoshida, Y., Yokoyama, K., Umemori, H., Inoue, A., Okabe, S., Manabe, T. & Yamamoto, T. (2003) p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N-methyl-d-aspartate receptor signaling. Mol Biol Cell, 14, 2921-2934.

Ni, J., Gao, Y., Gong, S., Guo, S., Hisamitsu, T. & Jiang, X. (2013) Regulation of mu-opioid type 1 receptors by microRNA134 in dorsal root ganglion neurons following peripheral inflammation. Eur J Pain, 17, 313-323.

Niederberger, E., Kynast, K., Lotsch, J. & Geisslinger, G. (2011) MicroRNAs as new players in the pain game. Pain, 152, 1455-1458.

Ogul, H., Umu, S.U., Tuncel, Y.Y. & Akkaya, M.S. (2011) A probabilistic approach to microRNA-target binding. Biochem Biophys Res Commun, 413, 111-115.

Okabe, T., Nakamura, T., Nishimura, Y.N., Kohu, K., Ohwada, S., Morishita, Y. & Akiyama, T. (2003) RICS, a novel GTPase-activating protein for Cdc42 and Rac1, is involved in the beta-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling. J Biol Chem, 278, 9920-9927.

Oleksiak, M.F., Churchill, G.A. & Crawford, D.L. (2002) Variation in gene expression within and among natural populations. Nat Genet, 32, 261-266.

Pan, Z., Zhu, L.J., Li, Y.Q., Hao, L.Y., Yin, C., Yang, J.X., Guo, Y., Zhang, S., Hua, L., Xue, Z.Y., Zhang, H. & Cao, J.L. (2014) Epigenetic modification of spinal miR-219 expression regulates chronic inflammation pain by targeting CaMKIIgamma. J Neurosci, 34, 9476-9483.

Paraskevopoulou, M.D., Georgakilas, G., Kostoulas, N., Vlachos, I.S., Vergoulis, T., Reczko, M., Filippidis, C., Dalamagas, T. & Hatzigeorgiou, A.G. (2013) DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows. Nucleic Acids Res, 41, W169-173.

Park, S.J., Cheon, E.J. & Kim, H.A. (2013) MicroRNA-558 regulates the expression of cyclooxygenase-2 and IL-1beta-induced catabolic effects in human articular chondrocytes. Osteoarthritis Cartilage, 21, 981-989.

Peltier, H.J. & Latham, G.J. (2008) Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA, 14, 844-852.

Poh, K.W., Yeo, J.F. & Ong, W.Y. (2011) MicroRNA changes in the mouse prefrontal cortex after inflammatory pain. Eur J Pain, 15, 801 e801-812.

Preall, J.B. & Sontheimer, E.J. (2005) RNAi: RISC gets loaded. Cell, 123, 543-545.

Radu, B.M., Bramanti, P., Osculati, F., Flonta, M.L., Radu, M., Bertini, G. & Fabene, P.F. (2013) Neurovascular unit in chronic pain. Mediators Inflamm, 2013, 648268.

Reid, R.W. & Fodor, A.A. (2008) Determining gene expression on a single pair of microarrays. BMC Bioinformatics, 9, 489.

Ruan, J.-P.P., Zhang, H.-X.X., Lu, X.-F.F., Liu, Y.-P.P. & Cao, J.-L.L. (2010) EphrinBs/EphBs signaling is involved in modulation of spinal nociceptive processing through a mitogen-activated protein kinases-dependent mechanism. Anesthesiology, 112, 1234-1249.

Santini, P., Politi, L., Vedova, P.D., Scandurra, R. & Scotto d'Abusco, A. (2014) The inflammatory circuitry of miR-149 as a pathological mechanism in osteoarthritis. Rheumatol Int, 34, 711-716.

Sauer, S.K., Bove, G.M., Averbeck, B. & Reeh, P.W. (1999) Rat peripheral nerve components release calcitonin gene-related peptide and prostaglandin E2 in response to noxious stimuli: evidence that nervi nervorum are nociceptors. Neuroscience, 92, 319-325.

Sayed, D. & Abdellatif, M. (2011) MicroRNAs in development and disease. Physiol Rev, 91, 827-887.

Schaible, H.G. (2007) Peripheral and central mechanisms of pain generation. Handb Exp Pharmacol, 3-28.

Schaible, H.G., Richter, F., Ebersberger, A., Boettger, M.K., Vanegas, H., Natura, G., Vazquez, E. & Segond von Banchet, G. (2009) Joint pain. Exp Brain Res, 196, 153-162.

Schaible, H.G., von Banchet, G.S., Boettger, M.K., Brauer, R., Gajda, M., Richter, F., Hensellek, S., Brenn, D. & Natura, G. (2010) The role of proinflammatory cytokines in the generation and maintenance of joint pain. Ann N Y Acad Sci, 1193, 60-69.

Serhan, C.N., Chiang, N. & Van Dyke, T.E. (2008) Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol, 8, 349-361.

Simon, G., Jianning, L., Sebastian, T., Simone, W., Wiebke, M., Julia, B., Khalad, K., Torben, R., Michaela, B., Daniel, V., Ari, W., Klaus-Armin, N. & Rohini, K. (2014) Oligodendrocyte ablation triggers central pain independently of innate or adaptive immune responses in mice. Nature Communications, 5, 5472

Souza, G.R., Talbot, J., Lotufo, C.M., Cunha, F.Q., Cunha, T.M. & Ferreira, S.H. (2013) Fractalkine mediates inflammatory pain through activation of satellite glial cells. Proc Natl Acad Sci U S A, 110, 11193-11198.

Stiban, J., Tidhar, R. & Futerman, A.H. (2010) Ceramide synthases: roles in cell physiology and signaling. Adv Exp Med Biol, 688, 60-71.

Strickland, E.R., Woller, S.A., Hook, M.A., Grau, J.W. & Miranda, R.C. (2014) The association between spinal cord trauma-sensitive miRNAs and pain sensitivity, and their regulation by morphine. Neurochem Int, 77, 40-49.

Tam Tam, S., Bastian, I., Zhou, X.F., Vander Hoek, M., Michael, M.Z., Gibbins, I.L. & Haberberger, R.V. (2011) MicroRNA-143 expression in dorsal root ganglion neurons. Cell Tissue Res, 346, 163-173.

Tan, P.H., Pao, Y.Y., Cheng, J.K., Hung, K.C. & Liu, C.C. (2013) MicroRNA-based therapy in pain medicine: Current progress and future prospects. Acta Anaesthesiol Taiwan, 51, 171-176.

Tan, P.H., Yang, L.C. & Ji, R.R. (2009) Therapeutic potential of RNA interference in pain medicine. Open Pain J, 2, 57-63.

van Iterson, M., Bervoets, S., de Meijer, E.J., Buermans, H.P., t Hoen, P.A., Menezes, R.X. & Boer, J.M. (2013) Integrated analysis of microRNA and mRNA expression: adding biological significance to microRNA target predictions. Nucleic Acids Res, 41, e146.

van Spronsen, M., van Battum, E.Y., Kuijpers, M., Vangoor, V.R., Rietman, M.L., Pothof, J., Gumy, L.F., van Ijcken, W.F., Akhmanova, A., Pasterkamp, R.J. & Hoogenraad, C.C. (2013) Developmental and Activity-Dependent miRNA Expression Profiling in Primary Hippocampal Neuron Cultures. PLoS One, 8, e74907.

Villa, G., Ceruti, S., Zanardelli, M., Magni, G., Jasmin, L., Ohara, P.T. & Abbracchio, M.P. (2010) Temporomandibular joint inflammation activates glial and immune cells in both the trigeminal ganglia and in the spinal trigeminal nucleus. Mol Pain, 6, 89.

Vo, N., Klein, M.E., Varlamova, O., Keller, D.M., Yamamoto, T., Goodman, R.H. & Impey, S. (2005) A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci U S A, 102, 16426-16431.

von Schack, D., Agostino, M.J., Murray, B.S., Li, Y., Reddy, P.S., Chen, J., Choe, S.E., Strassle, B.W., Li, C., Bates, B., Zhang, L., Hu, H., Kotnis, S., Bingham, B., Liu, W., Whiteside, G.T., Samad, T.A., Kennedy, J.D. & Ajit, S.K. (2011) Dynamic changes in the microRNA expression profile reveal multiple regulatory mechanisms in the spinal nerve ligation model of neuropathic pain. PLoS One, 6, e17670.

Voscopoulos, C. & Lema, M. (2010) When does acute pain become chronic? Br J Anaesth, 105 Suppl 1, i69-85.

Watkins, L.R., Milligan, E.D. & Maier, S.F. (2001) Glial activation: a driving force for pathological pain. Trends Neurosci, 24, 450-455.

Wibrand, K., Panja, D., Tiron, A., Ofte, M.L., Skaftnesmo, K.O., Lee, C.S., Pena, J.T., Tuschl, T. & Bramham, C.R. (2010) Differential regulation of mature and precursor microRNA expression by NMDA and metabotropic glutamate receptor activation during LTP in the adult dentate gyrus in vivo. Eur J Neurosci, 31, 636-645.

Willemen, H.L., Huo, X.J., Mao-Ying, Q.L., Zijlstra, J., Heijnen, C.J. & Kavelaars, A. (2012) MicroRNA-124 as a novel treatment for persistent hyperalgesia. J Neuroinflammation, 9, 143.

Winter, J., Jung, S., Keller, S., Gregory, R.I. & Diederichs, S. (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol, 11, 228-234.

Woolf, C.J. (2011) Central sensitization: implications for the diagnosis and treatment of pain. Pain, 152, S2-15.

Woolf, C.J. & Chong, M.S. (1993) Preemptive analgesia--treating postoperative pain by preventing the establishment of central sensitization. Anesth Analg, 77, 362-379.

Wu, D., Raafat, M., Pak, E., Hammond, S. & Murashov, A.K. (2011) MicroRNA machinery responds to peripheral nerve lesion in an injury-regulated pattern. Neuroscience, 190, 386-397.

Xu, J., Kang, Y., Liao, W.M. & Yu, L. (2012) MiR-194 regulates chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5. PLoS One, 7, e31861.

Zakharkin, S.O., Kim, K., Mehta, T., Chen, L., Barnes, S., Scheirer, K.E., Parrish, R.S., Allison, D.B. & Page, G.P. (2005) Sources of variation in Affymetrix microarray experiments. BMC Bioinformatics, 6, 214.

Zhao, J., Lee, M.C., Momin, A., Cendan, C.M., Shepherd, S.T., Baker, M.D., Asante, C., Bee, L., Bethry, A., Perkins, J.R., Nassar, M.A., Abrahamsen, B., Dickenson, A., Cobb, B.S., Merkenschlager, M. & Wood, J.N. (2010a) Small RNAs control sodium channel expression, nociceptor excitability, and pain thresholds. J Neurosci, 30, 10860-10871.

Zhao, X., He, X., Han, X., Yu, Y., Ye, F., Chen, Y., Hoang, T., Xu, X., Mi, Q.S., Xin, M., Wang, F., Appel, B. & Lu, Q.R. (2010b) MicroRNA-mediated control of oligodendrocyte differentiation. Neuron, 65, 612-626.