LncRNA Anxa10-203 enhances Mc1r mRNA stability to promote neuropathic pain by recruiting DHX30 in the trigeminal ganglion

Abstract

Background: Trigeminal nerve injury is one of the most serious complications in oral clinics, and the subsequent chronic orofacial pain is a consumptive disease. Increasing evidence demonstrates long non-coding RNAs (lncRNAs) play an important role in the pathological process of neuropathic pain. This study aims to explore the function and mechanism of LncRNA Anxa10-203 in the development of orofacial neuropathic pain.

Methods: A mouse model of orofacial neuropathic pain was established by chronic constriction injury of the infraorbital nerve (CCI-ION). The Von Frey test was applied to evaluate hypersensitivity of mice. RT-qPCR and/or Western Blot were performed to analyze the expression of Anxa10-203, DHX30, and MC1R. Cellular localization of target genes was verified by immunofluorescence and RNA fluorescence in situ hybridization. RNA pull-down and RNA immunoprecipitation were used to detect the interaction between the target molecules. Electrophysiology was employed to assess the intrinsic excitability of TG neurons (TGNs) in vitro.

Results: Anxa10-203 was upregulated in the TG of CCI-ION mice, and knockdown of Anxa10-203 relieved neuropathic pain. Structurally, Anxa10-203 was located in the cytoplasm of TGNs. Mechanistically, Mc1r expression was positively correlated with Anxa10-203 and was identified as the functional target of Anxa10-203. Besides, Anxa10-203 recruited RNA binding protein DHX30 and formed the Anxa10-203/DHX30 complex to enhance the stability of Mc1r mRNA, resulting in the upregulation of MC1R, which contributed to the enhancement of the intrinsic activity of TGNs in vitro and orofacial neuropathic pain in vivo.

Conclusions: LncRNA Anxa10-203 in the TG played an important role in orofacial neuropathic pain and mediated mechanical allodynia in CCI-ION mice by binding with DHX30 to upregulate MC1R expression.

Keywords: DExH-box helicase 30; Long non-coding RNA; Melanocortin 1 receptor; Neuropathic pain; RNA-binding proteins; Trigeminal ganglion.

Fig. 1

Anxa10-203 was up-regulated in the TG after CCI-ION. a Mechanical sensitivity threshold of ipsilateral whisker pad skin in Sham and CCI-ION mice. **P < 0.01, ***P < 0.001, n = 6/group. b, c The expression of the top 10 up-regulated (b) and 10 down-regulated (c) lncRNAs were verified in Sham and CCI-ION mice on day 7 after CCI-ION. *P < 0.05, ***P < 0.001, n = 7/group. d The expression of Anxa10-203 in the TG from mice of Sham and CCI-ION group at days 1, 3, 7, and 14. **P < 0.01, ***P < 0.001, n = 8/group. e The 50% WHT of the ipsilateral whisker pad region was increased in CCI-ION mice with Anxa10-203 silenced in the TG. *P < 0.05, ***P < 0.001, CCI+siAnxa10-203 versus Sham; ^&&&P < 0.001, CCI+siNC versus Sham; ^###P < 0.001, CCI+siAnxa10-203 versus CCI+siNC. n = 5/group. (f) The expression of Anxa10-203 decreased on day 4 after siAnxa10-203 injection. **P < 0.01, ***P < 0.001, n = 3/group

Fig. 2

Anxa10-203 was expressed in the cytoplasm of TGNs. a The distribution of Anxa10-203 was assessed by the subcellular fraction assay. U6 and Gapdh were positive controls for the nucleus and cytoplasmic expression gene respectively. b FISH and IF staining demonstrated that Anxa10-203 was mainly expressed in the cytoplasm of TGNs in vitro. Scare bar = 50 μm. c Anxa10-203 RNA was co-located with MAP2, IB4, and CGRP in vivo. Representative immunopositive cells were identified by white arrows. Scare bar = 50 μm

Fig. 3

DHX30 protein interacted with Anxa10-203 RNA. a Venn diagram of the proteins from the RNA pull-down-LCMS and RNA-protein interaction online algorithm (CatRAPID and RBPsuite). b Gene Ontology analysis showed the top 10 enriched biological process, cellular component, and molecular function of these overlapped proteins. c RNA-FISH and IF staining revealed the co-localization of DHX30 protein and Anxa10-203 RNA in the cytoplasm of TGNs. Scare bar = 50 μm. d RNA pull-down assay assessed Anxa10-203 RNA interacted with DHX30 protein, the antisense served as control. **P < 0.01, n = 3/group. e RIP assay further confirmed that DHX30 protein interacted with Anxa10-203 RNA in the TG of CCI-ION mice. *P < 0.05, n = 3/group. f The scores of Anxa10-203-DHX30 binding sites predicted by RBPsuite. g The Anxa10-203 sequence segments that interacted with DHX30 protein were evaluated by RNA pull-down

Fig. 4

DHX30 protein interacted with Mc1r mRNA. a Venn diagram of the RNAs from the RNA-protein interaction online algorithm CatRAPID and pain-related genes. b RIP was used to validate the interaction between DHX30 and the overlapped RNAs. *P < 0.05, n = 3/group. c RNA-FISH and IF staining revealed the co-localization of DHX30 protein and Mc1r mRNA in the cytoplasm of TGNs. Scare bar = 50 μm. d RNA pull-down assay assessed Mc1r mRNA interacted with DHX30, the antisense served as control. *P < 0.05, n = 3/group

Fig. 5

Anxa10-203 expression was associated with MC1R. a Mc1r expression in the TG on 1, 3, 7, and 14 days after CCI-ION. ***P < 0.001, n = 4/group. b Correlation analysis between Anxa10-203 and Mc1r in the TG by Pearson correlation analysis. R² = 0.7023, P < 0.0001, n = 20. c Anxa10-203 knockdown reversed the Mc1r mRNA increase induced by CCI-ION. *P < 0.05, **P < 0.01, n = 4/group. d, e Anxa10-203 knockdown reversed the MC1R protein increase induced by CCI-ION. *P < 0.05, n = 4/group. f Anxa10-203 was increased 7 days after LV-Anxa10-203 microinjection in naive mice. *P < 0.05, n = 3/group. g Mc1r mRNA was significantly increased in TG induced by Anxa10-203 over-expression. *P < 0.05, **P < 0.01, n = 3/group. h-i Anxa10-203 over-expression induced increased MC1R protein in naive mice. *P < 0.05, n = 3/group

Fig. 6

Anxa10-203 over-expression enhanced Mc1r mRNA stability through DHX30. a, b RNA and protein expression of DHX30 in Sham and CCI mice with or without DHX30 silenced. *P < 0.05, **P < 0.01, ***P<0.001, n = 4/group. c, d Expression of Mc1r in Sham and CCI-ION mice with or without DHX30 silenced was assessed by RT-qPCR (c) and WB (d). *P < 0.05, **P < 0.01, n = 3/group for RT-qPCR, n=4/group for WB. e, f The expression of DHX30 mRNA (e) and protein (f) were evaluated in the TGNs with DHX30 knockdown and Anxa10-203 over-expression in vitro. *P < 0.05, **P < 0.01, n = 4/group. g, h The expression of Mc1r mRNA (g) and protein (h) was evaluated in TGNs with DHX30 silenced and Anxa10-203 over-expressed in vitro. *P < 0.05, **P < 0.01, ***P < 0.001, n = 4/group for RT-qPCR, n=3/group for WB. i Representative images of nascent protein in TGNs of the two groups. Scare bar = 20 μm. j Quantitative analysis of fluorescence intensity of nascent protein in the two groups. n = 10/group. k The decay of Mc1r mRNA in different groups. *P < 0.05, ***P < 0.001, n = 4/group. oe: over-expression, n.s: no significance