Nuclear paraspeckle assembly transcript 1 promotes photophobia behavior in mice via miR-196a-5p/Trpm3 coupling

Abstract

Background: The long noncoding RNA, NEAT1, is recognized as a key regulator of proinflammatory gene expression; Yet, its functional role in migraine remains unexplored, despite the central role of neuroinflammatory mechanisms in migraine pathophysiology. This study examines the implication of NEAT1 in the trigeminal ganglion activation, which underlies photophobia associated with migraine.

Methods: Light aversion behavior was induced by intranasal injection of the TRPA1 activator, umbellulone. Male mouse behavior was assessed by the total time the mouse stays in the light between the dark and light compartments. To gain insight to the NEAT1-mediated photophobia mechanism, gene expression of candidate genes and non-coding RNAs interactions were assessed using RNA-sequencing, qPCR analysis, histology and dual-luciferase reporter gene assay.

Results: NEAT1 was upregulated in the trigeminal ganglion of male photophobia mice; Downregulation of NEAT1 by intravenous injection of shNEAT1 adeno-associated virus vectors attenuated NEAT1 expression and alleviated photophobia-like behavior in mice. The elevated NEAT1 expression in the trigeminal ganglion of photophobia mice corresponds to the downregulation of miR-196a-5p and upregulation Trpm3 RNA level. Predicted analysis suggested NEAT1/miR-196a-5p ceRNA network exists in photophobia mice. Indeed, knocking down NEAT1 upregulated miR-196a-5p, whilst downregulated Trpm3 gene expression level, in the trigeminal ganglion of photophobia mice. Further investigation using dual-luciferase reporter gene assay identified NEAT1 interacting with miR-196a-5p, whilst miR-196a-5p interacting with Trpm3. Similar to knocking down NEAT1, TRPM3 inhibition reduced photophobia-like behavior.

Conclusion: We conclude that NEAT1 is critical for promoting photophobia behavior via miR-196a-5p/Trpm3 coupling.

Keywords: Competing endogenous RNA; Light aversion; Migraine; NEAT1; Transient receptor potential melastatin 3; miR-196a-5p.

Fig. 1

RNA-sequencing analysis of TG revealed profound alternation of RNA levels of lncRNAs in the light aversion behavior mice. (A) The heatmap showed the levels of 125 altered lncRNAs in each sample of control (vehicle) and photophobia (UMB) group respectively. (B) The volcano plot depicted the numbers of downregulated and upregulated lncRNAs (|log2FoldChange|≥0, adjusted P-value ≤ 0.05) following photophobia. Each dot represents an RNA, and red and blue dots indicate down- and up-regulation, respectively. (C) Analysis of RNA-sequencing data to compare RNA levels of NEAT1 in the TG tissue of mice between the photophobia group and control group. (D) qPCR validation of NEAT1 RNA level in the TG of mice in photophobia group and control group, presented by the fold changes normalized to the geometric mean of the two reference genes (β-actin and PPIA). One-tailed unpaired t-test was used for comparisons among two independent groups. Significant differences were indicated by ns not significant, * P < 0.05

Fig. 2

Knockdown of NEAT1 alleviated C57BL/6J mouse light aversion behavior induced by intranasal injection of UMB. Light aversion was induced by intranasal injection (i.n.) of UMB (150 µg/kg). There was a total of three experimental groups: (1) photophobia (PBS, vehicle, i.v.), NEAT1 knockdown (2.5E + 13vg/kg AAV-shNEAT1, AAV, i.v.) and AAV negative control (2.5E + 13vg/kg AAV-NC, AAVNC, i.v.) groups. AAV-shNEAT1, AAV-NC and PBS were tail-vein injected 14 days prior to photophobia induction. Light aversion behaviour of each mouse was recorded before and after light aversion induction for self-comparison. (A-C) Total time in light (sec) of individual mouse before and after light aversion induction in the PBS (A), shNEAT1-AAV (B) and AAV-NC (C) groups. (D) Comparisons of total time in light (sec) after light aversion induction between the PBS and AAV, shNEAT1-AAV and AAV-NC groups. (E) Comparison of total time in light normalised to respective self-control among the PBS, shNEAT1-AAV, AAV and AAV-NC groups. (F) Representative images showing histological examination of AAV-infected cells in the TG of photophobia mice compared with the PBS control. (G) qPCR data on RNA levels of NEAT1 in the TG of three groups presented by the fold changes normalized to the geometric mean of the two reference genes (β-actin and PPIA). Blue indicates TG cell nucleus stained by DAPI; Green indicates cells infected by AAV vector. Group data were presented as mean ± SEM. One-tailed unpaired t-test was used for comparisons among two independent groups. Significant differences were indicated by ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. *Indicates comparison between dependent groups. ^#Indicates comparison between independent groups

Fig. 3

An upregulation ceRNA network modulated by NEAT1 was predicted in the TG of light aversion mice. (A) The heatmap showed the RNA level of 7 altered miRNAs in each sample of control (DMSO) and photophobia (UMB) groups. (B) The volcano plot depicted the numbers of 7 differentially expressed lncRNAs (|log2FoldChange|≥0, adjusted P-value ≤ 0.05) between UMB vs. DMSO groups, of which 4 downregulated, while 3 upregulated. (C) A Sankey Diagram illustrated the interactions of the RNAs among the upregulatory ceRNA network modulated by NEAT1 in the TG of light aversion mice. The expression level of Trpm3 (D), Rnf38 (E), Slc7a2 (F), Mir196a-1 (G) and Mir196a-2 (H) in TG of control and photophobia mice accessed by RNA sequencing analysis. Validation of the gene expression of Trpm3 (I), Rnf38 (J) and Slc7a2 (K) in the TG of mice in photophobia group and control group using qPCR. Group data were presented as mean ± SEM. One-tailed unpaired t-test was used for comparisons among two independent groups. Significant differences were indicated by ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001

Fig. 4

NEAT1 knockdown upregulated miR-196a-5p while downregulated Trpm3 following photophobia induction via miR-196a-5p/Trpm3 axis. Effects of NEAT1 knockdown on RNA levels of miR-196a-5p (A) and Trpm3 (B) in the TG of mice in the PBS, shNEAT1-AAV and AAV-NC groups following light aversion induction. In A and B, RNA levels were normalized to the geometric mean of β-actin and PPIA (Trpm3), U6 and 18 S (miR-196a-5p) and were represented. (C) Predicted miR-196a-5p binding sites in the region of NEAT1. (D) Predicted miR-196a-5p binding sites in the region of Trpm3. (E) RNA sequences of NEAT1 in human and mouse, which are highly conserved and possess a conserved miR-196a-5p–binding region. (F) RNA sequences of the 3′-UTRs of Trpm3 in human and mouse, which are highly conserved and possess a conserved miR-196a-5p–binding region. (G) Dual-luciferase reporter assay verifying the interaction between the 3′-UTR of NEAT1 and miR-196a-5p. Data are representative of 6 independent experiments. (H) Dual-luciferase reporter assay verifying the interaction between the 3′-UTR of Trpm3 and miR-196a-5p. Data are representative of 6 independent experiments. The fold changes in qPCR were normalized to the geometric mean of β-actin and PPIA(Trpm3), U6 and 18 S(miR-196a-5p) and RNA levels were determined using 2^−ΔCT method. NC, negative control; WT, wild-type; Mut, mutant. Group data were presented as mean ± SEM. One-tailed unpaired t-test was used for comparisons among two independent groups. Significant differences were indicated by ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. *Indicates comparison between dependent groups. ^#Indicates comparison between independent groups

Fig. 5

Inhibition of TRPM3 reversed the photophobia behavior of C57BL/6J induced by intranasal injection of UMB. Light aversion was induced by intranasal injection (i.n.) of UMB (150 µg/kg). 0.2% DMSO was injected as the model control. There was a total of three experimental groups: control group (vehicle control, i.p. 0.25% DMSO.), photophobia group in the absence (i.p., 0.25% DMSO) or presence of the TRPM3 inhibitor, ISO (i.p.,10 mg/kg). ISO or its vehicle was intraperitoneally injected for 2 consecutive days prior to photophobia induction. (A-C) Comparison of total time in light (sec) before and after light aversion induction in the control (A), photophobia (B) and photophobia + ISO (C) groups. (D) Effects of ISO on light avoidance before and after light aversion induction by comparing total time in light (sec) between the control and photophobia, or photophobia and ISO groups. (E) Effects of ISO on total time in light (normalised to self-control) following light aversion induction. Group data were presented as mean ± SEM. One-tailed, unpaired t-test was used for comparisons between two independent groups. Significant differences were indicated by ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001; ^#P < 0.05, ^##P < 0.01. *Indicates comparison between dependent groups. ^#Indicates comparison between independent groups

Fig. 6

Schematic representation of the proposed regulatory mechanism of NEAT1 in UMB-induced light aversion via miR-196a-5p/Trpm3 axis. Triggers of photophobia, such as UMB, upregulate gene expression of NEAT1, which enhances Trpm3 gene expression by inhibiting miR-196a-5p expression and activity through competing endogenous RNA mechanism. This upregulation of Trpm3 gene expression may lead to the production of the TRPM3 protein, whose activation sustains trigeminovascular sensitization via calcium influx and neuroinflammation, ultimately affecting photophobia behavior. The figure was created in BioRender. Lab, M. (2025). https://BioRender.com/bem83j5. TRPA1, Transient receptor potential ankyrin type 1. TRPM3: Transient receptor potential melastatin-3