Regulation Of μ-opioid Type1Receptors By MicroRMA134in Dorsal Root Ganglion Neurons Following Peripheral Inflammation

Part Ⅰ Study on relationship of microRNA134and MOR1indorsal root ganglion neurons after inflammatory painObjective MOR1is the main transcript of μ opioid receptor (MOR） gene, whichrepresents a mandatory molecule for the analgesic effects of opioids and plays animportant role in the pathology of inflammatory pain. By using bioinformation analysis, anexact match to the seed sequence of miR-134was found in3’-untranslated region of MOR1.Given the important role of MOR1in pain modulation, the purpose of this study is toinvestigate whether miR-134can regulate the MOR1following allodynia.Methods Using the Freund’s adjuvant (CFA）-induced chronic inflammatory painmodel, we investigates the expression profiles of miR-134and MOR1in male Wistar ratdorsal root ganglia (DRG） using quantitative real-time PCR at time points of4h,1d,4d,7d and14d. The relationship of miR-134and MOR1expressions was analyzed by linearregression. The expression of MOR1was examined also by immunofluorescencedetection in ipsilateral L5DRG of inflammatory pain rats at the time of4h,4d and14d.Finally, we further examine the expression of MOR1combined with that of miRNA134inthe same DRGs by in situ hybridization and immunohistochemistry.Results The pressure stimulus threshold and thermal stimulus latency of ipsilateralfoot were extended after CFA injection in Wistar rats. Compared with the control group,MOR1mRNA levels of experimental group significantly increased at4h following CFAinjection（p＜0.01）, with the maximum on day4after induction of inflammation （p＜0.01）, correlating with the development of thermal and pressure stimulus threshold after CFA injection. Conversely, the expression level of miRNA134was gradually decreasedfrom1d to4d(p＜0.05）and then gradually increased from7d to14d (p＜0.01）. Linearregression analysis showed that miR-134expression level was inversely related withMOR1expression level (R2=0.7363, p＜0.001）. Immunofluorescence showed that MOR1immunoreactivity was predominantly found in cytoplasm of neurons in rat DRG neurons,especially in those small diameter neurons. The percentage of MOR1-positive neuronssignificantly increased after4h of CFA injection, and reached the maximal at4d. Theimmunofluorescence intensity detection for MOR1showed the similar results that intensityincreased after CFA injection and raised to the top after4days. Down-regulation ofmiRNA134and upregulation of MOR1in the same DRG tissues after inflammatory painwere observed using the method of in situ hybridization and immunohistochemistry. Thechanges were detected mainly in small diameter neurons.Conclusions Our present data suggested a model that miR-134participated inCFA-induced inflammatory pain by balancing the expression of MOR1in DRGs. Theseresults implied that miR-134might be a potential target for the treatment of neuropathicpain.Part Ⅱ Study on relationship of microRNA134and MOR1inSH-SY5Y cell line after transfectionObjective MicroRNAs are non-coding molecules that primarily regulate geneexpression at the post-transcriptional level by predominantly hybridizing tocomplementary sequences in the3’-UTR of their corresponding mRNAs. Considering theimportant roles of MOR1in pain modulation and the relationship of miRNA134andMOR1, we study in vivo in part1. The purpose of this study is to detect whethermiRNA134can regulate the MOR1in vitro.Methods In this study, we transfected LNA-anti-miRNA134(10nM、50nM、100nM）into SH-SY5Y cell line using lipofectamine2000. We investigated the expression ofmiRNA134and MOR1mRNA after transfection by the way of real-time PCR.MOR1 (protein）expression in cell culture after transfection was detected by Western Blot,immunohistochemistry and electron microscopy.Results After transfection with LNA-anti-miRNA134(10nM,50nM,100nM） usinglipofectamine2000for48h, compared with NC group, the expression of miRNA134wassignificantly reduced（p＜0.01）, while the level of MOR1mRNA increased（p＜0.01）.They changed gradually according to the density of LNA-anti-miRNA134. After treatedfor72h, the expression of MOR1increased accordingly by western blot, as same as theresult of real-time PCR（p＜0.01）. We found the expression of MOR1increased afterLNA-anti-miRNA134(100nM） transfection by the methods of immunohistochemistry.The percentage of MOR1-positive neurons significantly increased after transfection. Theimmunofluorescence detection for MOR1showed the similar results that the intensityincreased after transfection of LNA-anti-miRNA134. We also found the increased MOR1after transfection at the level of ultra microstructure by using electron microscopy analysis.Conclusions miRNA134can inversely regulate MOR1in vitro. Inhibition ofmiRNA134activity leads to an increase of the expression of MOR1and MOR1mRNA,which can also testify the relationship of miRNA134and MOR1partly in vivo.MiRNA134is an important potential target in pain modulation.