PMID- 36202171 OWN - NLM STAT- MEDLINE DCOM- 20221215 LR - 20230208 IS - 1090-2139 (Electronic) IS - 0889-1591 (Linking) VI - 107 DP - 2023 Jan TI - CircNf1-mediated CXCL12 expression in the spinal cord contributes to morphine analgesic tolerance. PG - 140-151 LID - S0889-1591(22)00400-7 [pii] LID - 10.1016/j.bbi.2022.09.018 [doi] AB - BACKGROUND: Severe pain in patients can be alleviated by morphine treatment. However, long-term morphine treatment induces analgesic tolerance and the molecular mechanism of morphine analgesic intolerance is still not fully elucidated. Therefore, a novel target for improving morphine analgesic tolerance is required. Whole-genome sequencing showed that circNf1 is highly expressed in the dorsal horns of morphine-treated rats. Circular RNAs (circRNAs) are known to be unique and conserved cellular molecules that are mostly present in cytoplasm and participate in various biochemical processes with different functions. Therefore, we focused on exploring the molecular mechanism by which circNf1 contributes to morphine analgesic tolerance. METHODS: CircRNA sequencing revealed differential expression of circRNAs after morphine treatment, and bioinformatics software programs (miRNAda, PicTar, and RNAhybrid) were used to predict possible mRNAs and binding sites. RNA binding protein immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), fluorescence in situ hybridization (FISH), western blotting, biotin-coupled probe pull-down assay, luciferase assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to detect and measure the expression levels of circRNAs, mRNAs, and proteins. Intrathecal injections of small interfering RNAs (siRNAs), microRNA (miRNA) agomirs, and functional virus microinjections were administered to artificially mediate the expression of molecules. Tail immersion and hotplate tests were performed to evaluate morphine analgesic tolerance. RESULTS: Morphine-induced circNf1 expression was high in the spinal cord. RIP-PCR and luciferase assay data showed that circNf1 could combine with both miR-330-3p and miR-665, and FISH showed that circNf1 co-localized with miR-330-3p and miR-665. qRT-PCR assay showed downregulation of miR-330-3p and miR-665 in morphine-treated rats; western blotting results showed that CXCL12 increased after morphine treatment, however, the upregulation of CXCL12 could be alleviated after the intrathecal injection of miR-330-3p as well as miR-665 agomir. qRT-PCR indicated that circNf1 can bind to CXCL12 promoter, the increased circNf1 can enhance CXCL12 mRNA in naive rats, and inhibition of circNf1 can alleviate the upregulation of CXCL12 mRNA in morphine-treated rats. Behavioral tests revealed that inhibition of circNf1 and CXCL12 and the enhancement of miR-330-3p and miR-665 can alleviate morphine analgesic tolerance. CONCLUSIONS: Our study indicates a novel pathway that can contribute to morphine analgesic tolerance, the circRNA to cytokine pathway, in which circNf1 functions as a sponge for miR-330-3p and miR-665 and induces the upregulation of CXCL12 at both transcriptional and translational levels in morphine-treated rats. CI - Copyright (c) 2022 Elsevier Inc. All rights reserved. FAU - Bai, Xiaohui AU - Bai X AD - Department of Anesthesiology, Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation. Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. FAU - Huang, Yongtian AU - Huang Y AD - Department of Anesthesiology, State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China. FAU - Zhang, Kun AU - Zhang K AD - Department of Anesthesiology, State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China. FAU - Huang, Wan AU - Huang W AD - Department of Anesthesiology, State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China. FAU - Mu, Yanyu AU - Mu Y AD - Department of Anesthesiology, State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China. FAU - Li, Yujuan AU - Li Y AD - Department of Anesthesiology, Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation. Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. Electronic address: liyuj@mail.sysu.edu.cn. FAU - Ouyang, Handong AU - Ouyang H AD - Department of Anesthesiology, State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China. Electronic address: ouyhd@sysucc.org.cn. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20221003 PL - Netherlands TA - Brain Behav Immun JT - Brain, behavior, and immunity JID - 8800478 RN - 76I7G6D29C (Morphine) RN - 0 (RNA, Messenger) RN - 0 (CXCL12 protein, rat) RN - 0 (Chemokine CXCL12) RN - 0 (MIRN665 microRNA, rat) RN - 0 (MicroRNAs) SB - IM MH - Rats MH - Animals MH - *Morphine/pharmacology MH - In Situ Hybridization, Fluorescence MH - Spinal Cord MH - RNA, Messenger MH - Chemokine CXCL12 MH - *MicroRNAs/genetics OTO - NOTNLM OT - CXCL12 OT - Circular RNAs OT - Morphine tolerance, Neuroinflammation OT - miRNA sponge COIS- Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2022/10/07 06:00 MHDA- 2022/12/15 06:00 CRDT- 2022/10/06 19:13 PHST- 2022/05/21 00:00 [received] PHST- 2022/09/20 00:00 [revised] PHST- 2022/09/30 00:00 [accepted] PHST- 2022/10/07 06:00 [pubmed] PHST- 2022/12/15 06:00 [medline] PHST- 2022/10/06 19:13 [entrez] AID - S0889-1591(22)00400-7 [pii] AID - 10.1016/j.bbi.2022.09.018 [doi] PST - ppublish SO - Brain Behav Immun. 2023 Jan;107:140-151. doi: 10.1016/j.bbi.2022.09.018. Epub 2022 Oct 3.