MicroRNA-1224 Splicing CircularRNA-Filip1l in an Ago2-Dependent Manner Regulates Chronic Inflammatory Pain via Targeting Ubr5.

Dysfunctions of gene transcription and translation in the nociceptive pathways play the critical role in development and maintenance of chronic pain. Circular RNAs (circRNAs) are emerging as new players in regulation of gene expression, but whether and how circRNAs are involved in chronic pain remain elusive. We showed here that complete Freund's adjuvant-induced chronic inflammation pain significantly increased circRNA-Filip1l (filamin A interacting protein 1-like) expression in spinal neurons of mice. Blockage of this increase attenuated complete Freund's adjuvant-induced nociceptive behaviors, and overexpression of spinal circRNA-Filip1l in naive mice mimicked the nociceptive behaviors as evidenced by decreased thermal and mechanical nociceptive threshold. Furthermore, we found that mature circRNA-Filip1l expression was negatively regulated by miRNA-1224 via binding and splicing of precursor of circRNA-Filip1l (pre-circRNA-Filip1l) in the Argonaute-2 (Ago2)-dependent manner. Increase of spinal circRNA-Filip1l expression resulted from the decrease of miRNA-1224 expression under chronic inflammation pain state. miRNA-1224 knockdown or Ago2 overexpression induced nociceptive behaviors in naive mice, which was prevented by the knockdown of spinal circRNA-Filip1l. Finally, we demonstrated that a ubiquitin protein ligase E3 component n-recognin 5 (Ubr5), validated as a target of circRNA-Filip1l, plays a pivotal role in regulation of nociception by spinal circRNA-Filip1l. These data suggest that miRNA-1224-mediated and Ago2-dependent modulation of spinal circRNA-Filip1l expression regulates nociception via targeting Ubr5, revealing a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.SIGNIFICANCE STATEMENT circRNAs are emerging as new players in regulation of gene expression. Here, we found that the increase of circRNA-Filip1l mediated by miRNA-1224 in an Ago2-dependent way in the spinal cord is involved in regulation of nociception via targeting Ubr5 Our study reveals a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.

Hydroxymethylation of microRNA-365-3p Regulates Nociceptive Behaviors via Kcnh2.

DNA 5-hydroxylmethylcytosine (5hmC) catalyzed by ten-eleven translocation methylcytosine dioxygenase (TET) occurs abundantly in neurons of mammals. However, the in vivo causal link between TET dysregulation and nociceptive modulation has not been established. Here, we found that spinal TET1 and TET3 were significantly increased in the model of formalin-induced acute inflammatory pain, which was accompanied with the augment of genome-wide 5hmC content in spinal cord. Knockdown of spinal TET1 or TET3 alleviated the formalin-induced nociceptive behavior and overexpression of spinal TET1 or TET3 in naive mice produced pain-like behavior as evidenced by decreased thermal pain threshold. Furthermore, we found that TET1 or TET3 regulated the nociceptive behavior by targeting microRNA-365-3p (miR-365-3p). Formalin increased 5hmC in the miR-365-3p promoter, which was inhibited by knockdown of TET1 or TET3 and mimicked by overexpression of TET1 or TET3 in naive mice. Nociceptive behavior induced by formalin or overexpression of spinal TET1 or TET3 could be prevented by downregulation of miR-365-3p, and mimicked by overexpression of spinal miR-365-3p. Finally, we demonstrated that a potassium channel, voltage-gated eag-related subfamily H member 2 (Kcnh2), validated as a target of miR-365-3p, played a critical role in nociceptive modulation by spinal TET or miR-365-3p. Together, we concluded that TET-mediated hydroxymethylation of miR-365-3p regulates nociceptive behavior via Kcnh2. SIGNIFICANCE STATEMENT: Mounting evidence indicates that epigenetic modifications in the nociceptive pathway contribute to pain processes and analgesia response. Here, we found that the increase of 5hmC content mediated by TET1 or TET3 in miR-365-3p promoter in the spinal cord is involved in nociceptive modulation through targeting a potassium channel, Kcnh2. Our study reveals a new epigenetic mechanism underlying nociceptive information processing, which may be a novel target for development of antinociceptive drugs.

DNA Hydroxymethylation by Ten-eleven Translocation Methylcytosine Dioxygenase 1 and 3 Regulates Nociceptive Sensitization in a Chronic Inflammatory Pain Model.

BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase converts 5-methylcytosine in DNA to 5-hydroxymethylcytosine, which plays an important role in gene transcription. Although 5-hydroxymethylcytosine is enriched in mammalian neurons, its regulatory function in nociceptive information processing is unknown. METHODS: The global levels of 5-hydroxymethylcytosine and ten-eleven translocation methylcytosine dioxygenase were measured in spinal cords in mice treated with complete Freund's adjuvant. Immunoblotting, immunohistochemistry, and behavioral tests were used to explore the downstream ten-eleven translocation methylcytosine dioxygenase-dependent signaling pathway. RESULTS: Complete Freund's adjuvant-induced nociception increased the mean levels (± SD) of spinal 5-hydroxymethylcytosine (178 ± 34 vs. 100 ± 21; P = 0.0019), ten-eleven translocation methylcytosine dioxygenase-1 (0.52 ± 0.11 vs. 0.36 ± 0.064; P = 0.0088), and ten-eleven translocation methylcytosine dioxygenase-3 (0.61 ± 0.13 vs. 0.39 ± 0.08; P = 0.0083) compared with levels in control mice (n = 6/group). The knockdown of ten-eleven translocation methylcytosine dioxygenase-1 or ten-eleven translocation methylcytosine dioxygenase-3 alleviated thermal hyperalgesia and mechanical allodynia, whereas overexpression cytosinethem in naïve mice (n = 6/group). Down-regulation of spinal ten-eleven translocation methylcytosine dioxygenase-1 and ten-eleven translocation methylcytosine dioxygenase-3 also reversed the increases in Fos expression (123 ± 26 vs. 294 ± 6; P = 0.0031; and 140 ± 21 vs. 294 ± 60; P = 0.0043, respectively; n = 6/group), 5-hydroxymethylcytosine levels in the Stat3 promoter (75 ± 16.1 vs. 156 ± 28.9; P = 0.0043; and 91 ± 19.1 vs. 156 ± 28.9; P = 0.0066, respectively; n = 5/group), and consequent Stat3 expression (93 ± 19.6 vs. 137 ± 27.5; P = 0.035; and 72 ± 15.2 vs. 137 ± 27.5; P = 0.0028, respectively; n = 5/group) in complete Freund's adjuvant-treated mice. CONCLUSIONS: This study reveals a novel epigenetic mechanism for ten-eleven translocation methylcytosine dioxygenase-1 and ten-eleven translocation methylcytosine dioxygenase-3 in the modulation of spinal nociceptive information via targeting of Stat3.

Using cross efficiency method integrating regret theory and WASPAS to evaluate road safety performance of Chinese provinces.

Currently, the road safety performance of a national- or province-level is absorbing much attention from practitioners and academics. A variety of models based on Data Envelopment Analysis (DEA) have been presented to appraise road safety performance in previous literature. However, the main weakness of these methods is that the decision-maker's psychological behavior is neglected. Besides, Cross Efficiency Method (CEM) adopts the weighted average to aggregate the cross efficiencies which may lead to information loss. To address these limitations, a hybrid road safety evaluation model was constructed by integrating CEM, regret theory, and Weighted Aggregated Sum Product Assessment (WASPAS) to appraise the road safety performance of Chinese provinces. In the presented model, regret theory is employed to depict the psychological behavior of the decision-maker. Then entropy weight method is utilized to calculate the weight of self-evaluation and peer-evaluation. To reduce information loss, WASPAS is adopted to fuse the cross efficiencies and derive the ranking of decision-making units. Finally, the presented model is employed to assess the road safety efficiency in the province of mainland China. The results show that the mean of road safety efficiency score for the eastern, central, and western areas is gradually decreasing. Furthermore, the outcomes of a comparative analysis represent that the results obtained by the proposed model outperform the CEM and DEA road safety model.