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

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