LncRNA 4930544M13Rik-201 regulates CACNA2D1 expression via interacting with hnRNPA2B1 to promote neuropathic pain following nerve injury

doi:10.21203/rs.3.rs-4685834/v1

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LncRNA 4930544M13Rik-201 regulates CACNA2D1 expression via interacting with hnRNPA2B1 to promote neuropathic pain following nerve injury

https://doi.org/10.21203/rs.3.rs-4685834/v1

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Long non-coding RNAs (LncRNAs) have recently been reported to play a crucial role in neuropathic pain (NP) resulting from peripheral nerve injury. However, the underlying mechanisms are not fully elucidated. Here, we investigated the role and mechanism of lncRNA 4930544M13Rik-201, a significantly up-regulated lncRNA in both trigeminal ganglion (TG) and dorsal root ganglion (DRG) following peripheral nerve injury, as determined by previous RNA-sequencing results, in the pathogenesis of trigeminal NP induced by infraorbital nerve chronic constriction injury (CCI-ION) in mice. LncRNA 4930544M13Rik-201 was predominantly located in the nuclei of neurons and significantly upregulated in the TG after CCI-ION. Silencing the expression of 4930544M13Rik-201 alleviated mechanical allodynia induced by CCI-ION, while over-expression of 4930544M13Rik-201 in the TG of the WT mice caused orofacial allodynia. Moreover, calcium voltage-gated channel auxiliary subunit alpha 2 delta 1 (CACNA2D1) was identified as the downstream target of lncRNA 4930544M13Rik-201. Notably, 4930544M13Rik-201 increased the stabilization of Cacna2d1 mRNA and protein expression via interacting with heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1). Furthermore, inhibition of CACNA2D1 and silencing of hnRNPA2B1 both alleviated the allodynia induced by CCI-ION and the overexpression of 4930544M13Rik-201. Taken together, these results suggest that 4930544M13Rik-201 plays a critical role in the regulation of trigeminal NP induced by CCI-ION through upregulating Cacna2d1 expression via binding to hnRNPA2B1. These findings have important implications for the development of new therapeutic strategies for the treatment of NP by targeting the 4930544M13Rik-201—hnRNPA2B1—CACNA2D1 axis.

lncRNA 4930544M13Rik-201

hnRNPA2B1

CACNA2D1

neuropathic pain

nerve injury

Neuropathic pain (NP) is a chronic pain condition that arises from injury or disease to the peripheral nerve or central nervous system [1]. NP is characterized by allodynia, hyperalgesia, and spontaneous pain, which affects the quality of life for 7–10% of the population in the world [2,3]. Nevertheless, current pharmacotherapy for NP including anticonvulsant medications, tricyclic antidepressants, serotonin noradrenaline reuptake inhibitors, as well as topical medications such as capsaicin and lidocaine are not always effective in the long term [4,5], leaving many patients with poor quality of life [6]. Therefore, it is crucial and urgent to understand the underlying mechanism of NP and identify potential targets for effective therapeutic interventions.

Long non-coding RNAs (lncRNAs) are categorized as RNA molecules exceeding 200 nucleotides in length, lacking or having minimal protein-coding potential [7]. LncRNAs can affect specific gene expression through various mechanisms, such as protein-lncRNA interaction [8], post-translational modification [9], and epigenetic regulation [10], thus participating in the modulation of the pathological process of human diseases. Clinical studies have revealed that specific lncRNAs are dynamically regulated in NP states [11]. For instance, the expression of lncRNA NONRATT021972 is significantly elevated and associated with NP of type 2 diabetes neuropathy patients [11]. In addition, the intrathecal injection of antisense oligonucleotides targeting lncRNA can significantly alleviate somatic NP caused by spinal cord injury [12]. To explore the role of lncRNA in NP of the trigeminal nervous system, we previously conducted a whole transcriptome sequencing of TGs from mice with trigeminal nerve injury, and lncRNA 4930544M13Rik-201 was found to be significantly upregulated after nerve injury [13]. Interestingly, obvious upregulation of lncRNA 4930544M13Rik-201 in the dorsal root ganglions (DRGs) of NP models was also observed by RNA-seq analyses [14–16]. However, the functional role of lncRNA 4930544M13Rik-201 and its mechanism underlying NP remain to be further determined.

CACNA2D1 (calcium voltage-gated channel auxiliary subunit alpha2 delta1, α₂δ-1), a subunit of voltage-activated calcium channel (VGCC), has been previously demonstrated to play an important role in the development of NP [3]. For instance, previous studies have reported a significant elevation in CACNA2D1 expression in the DRG and spinal cord following peripheral nerve injury [17,18]. Moreover, ablation of Cacna2d1 mRNA alleviated mechanical hypersensitivity following nerve injury [19]. Notably, in our prior investigations, we observed a significant upregulation of CACNA2D1 within the TG after inferior alveolar nerve injury, resulting in trigeminal NP. Besides, symptom relief of ectopic pain was achieved through the intra-TG injection of the CACNA2D1 inhibitor [17]. However, the mechanism underlying the up-regulation of CACNA2D1 in the peripheral ganglion after nerve injury remains unclear.

In this study, with the trigeminal NP model built by chronic constriction injury of infraorbital nerve (CCI-ION), we have made an innovative discovery that lncRNA 4930544M13Rik-201 could regulate the expression of CACNA2D1 via interacting with heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), which played an important role in the NP following trigeminal nerve injury. These findings suggest that lncRNA 4930544M13Rik-201 may be a novel therapeutic target for treating NP.

2.1 Animals and neuropathic pain model

C57BL/6 mice (Male and female, 8–10 weeks) were purchased from the Laboratory Animal Center of Sichuan University. The mice were housed in a controlled environment, including a controlled temperature (22–24°C), and a 12-hour light-dark cycle, with freely available food and water. After acclimatization for 5–7 days, the mice were randomly divided into experimental groups. This study was approved by the Ethics Committee of West China College of Stomatology, Sichuan University (Approval No. WCHSIRB-D-2021-564). All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the International Association for the Study of Pain [20].

The CCI-ION model of mice was used to induce trigeminal NP [21,22]. Briefly, the mice were anesthetized with 2.5% tribromoethyl alcohol (Sigma-Aldrich, USA, 250 mg/kg, i.p.), and a 2 mm incision was made in the left palatal buccal mucosa, then the infraorbital nerve was exposed and tied for twice with three 5 − 0 chromic gut ligatures. The incision was closed with 6 − 0 nylon sutures. Mice in the sham group underwent the same surgical procedure on the left side without ligation of the infraorbital nerve.

2.2 Behavioral test

To evaluate orofacial mechanical hypersensitivity in mice, the head withdrawal threshold (HWT) was determined by a series of von Frey filaments (0.04 g, 0.16 g, 0.40 g, 0.60 g, 1.00 g, 1.40 g, 2.00 g, 4 .00g, Stoelting Company, Wood Dale, USA) following a blinded approach, as previously outlined by [23,24]. Briefly, mice were accustomed to the experimental procedure for 5 days before surgical treatments and had the vibrissae carefully shaved 1 day before the von Frey filament test. During the behavioral testing, mice were gently placed within a metal mesh cage (8 cm × 8 cm 8 cm) without activity restriction. After a 30-minute acclimation, filaments were presented to the ION-innervated area at an interval of 5 minutes, according to the up-down rules [23]. The 50% HWT was calculated and determined as a marker of the mechanical withdrawal threshold. The behavioral testing was conducted under blinded conditions.

2.3 Administration of drugs into the TG

In line with our prior investigations [25], drug administration into the trigeminal ganglion (TG) was carried out utilizing a stereotaxic apparatus (RWD, China). After anesthesia induction, mice were securely positioned on the apparatus. A 15 mm incision was made to expose the anterior fontanelle and the surrounding cranial bone. The anterior fontanelle was marked as the reference point for localization. Subsequently, the apparatus was adjusted to mark the cranial bone at a point located 1 mm to the left of the anterior fontanelle and 4 mm posterior to it. A micro-syringe (Hamilton, China) was then inserted vertically at the marked position to a depth of 6.5 mm, followed by a slow injection of drugs over a 2-minute duration. The micro-syringe was gradually withdrawn after remaining in place for 5 minutes.

The administration of the following drugs was carried out following the manufacturer's instructions or established protocols from prior studies: 1 µg siRNA of 4930544M13Rik-201 or negative control (NC) were synthesized by Genepharma (Shanghai, China) and diluted with 0.5 µl Entranster in vivo RNA reagent (Engreen Biosystem, China) and 1.5 µl saline solution; 1 µg pcDNA3.1-4930544M13Rik-201 (pcDNA3.1-lnc) full-length plasmid or pcDNA3.1 plasmid vector was diluted with Entranster in vivo DNA reagent (Engreen Biosystem, China); Gabapentin (GBP, a ligand inhibitor of CACNA2D1, Abmole, USA) was prepared as previously documented [26]. Sequences of siRNAs are listed in Table S1.

2.4 Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

Mice were anesthetized by 2.5% tribromoethyl alcohol, and then the ipsilateral TG was quickly isolated and immediately restored in RNA preservation solution (Servicebio, China). Total RNA was extracted from homogenized TG tissue using the RNA Extraction Kits (Takara, Japan) according to the manufacturer’s instructions. The RNA concentration was determined using a NanoDrop spectrometer (Thermofisher, USA). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the Prime Script^™ RT Reagent Kit (Takara, Japan). The resulting cDNA was then used as a template for quantitative PCR (qPCR) using the TB Green Premix Ex Taq II (Takara, Japan) and gene-specific primers listed in Table S2. The qPCR was performed on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, USA) with the following cycling conditions: 1 cycle of 95℃ for 30 s, 40 cycles: 95℃ for 5 s, 60℃ for 30 s. Gene expression levels were calculated using the 2^^-ΔΔCt method, normalized to the housekeeping gene Gapdh.

2.5 Western blotting (WB)

Proteins were extracted from homogenized mice TG tissue using RIPA lysis buffer (Beyotime, China) via mechanical disruption. The protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein (20 µg) were separated on a 10% SDS-PAGE gel (Epizyme, China) and transferred onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk in TBST buffer (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature and then incubated with primary antibody overnight at 4°C. The following primary antibodies were used: anti-CACNA2D1 (Abcam, Ab253190, UK), anti-hnRNPA2B1 (Proteintech, 14813-1-AP, China) and anti-α-Tubulin (Genetex, GTX112141, USA). After washing with TBST, the membrane was incubated with an anti-rabbit HRP-conjugated secondary antibody (SAB, 3012, USA) for 1 h at room temperature. The secondary antibody was an anti-rabbit IgG HRP-linked antibody. The protein bands were visualized using an ECL Western Blotting Detection System (Thermofisher, USA) and analyzed using ImageJ software. The protein expression levels were normalized to α-Tubulin and presented as fold change relative to control samples.

2.6 RNA pull-down

To investigate proteins interacting with 4930544M13Rik-201 in the TG of CCI-ION mice, an RNA pull-down assay was conducted. Briefly, the probes were synthesized using T7 RNA Polymerase Mix (Vazyme, China), and then labeled by Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermofisher, USA). Then the probes were incubated with TG protein extracts, the RNA-protein complexes were captured by using Streptavidin-coated magnetic beads (Thermofisher, USA). After thorough washing, the binding proteins were eluted and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) or western blotting to identify potential interacting partners. LC-MS/MS was performed by Sangon Biotech (Shanghai, China).

2.7 RNA immunoprecipitation (RIP)

To identify RNA fragments that interact with hnRNPA2B1 in vivo, RNA immunoprecipitation (RIP) was performed using an RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). Briefly, the TG tissues of CCI-ION mice were collected and lysed into single-cell suspension, then the lysates were incubated with hnRNPA2B1 antibody (Proteintech, 14813-1-AP, China). After antibody-RNA-protein complexes were formed, the Protein A/G magnetic beads were added to the mixture, facilitating the binding of the immune complexes to the beads. The beads were washed with washing buffer to eliminate non-specific binding. Proteinase K treatment was applied to remove proteins, and then the isolated RNA underwent reverse transcription, followed by RT-qPCR to quantify the abundance of 4930544M13Rik-201 and Cacna2d1 mRNA in the immunoprecipitated complexes.

2.8 Immunofluorescence (IF)

CCI-ION mice were deeply anesthetized with 2.5% tribromoethyl alcohol. The TG tissues were quickly removed and immediately incubated with 4% paraformaldehyde and equilibrated in 30% sucrose overnight. Then the frozen sections (8 µm) of TG tissue were cut by a cryostat (Leica, Germany) and mounted on glass slides. The sections were stored at -20℃ for subsequent assessments. For visualizing target protein expression patterns in the TG, the prepared sections were incubated with 0.25% Triton X-100 for 15min, blocking by 10% goat serum for 30 min. Primary antibodies targeting specific proteins were applied and incubated overnight at 4℃. After thorough washing, the sections were incubated with corresponding fluorophore-conjugated secondary antibodies. DAPI counterstaining facilitated nuclear identification, anti-fading mounting medium were applied to maintain fluorescence intensity for subsequent imaging. All primary and secondary antibodies in this study are listed in Table S3. Images were captured utilizing a confocal laser scanning microscope (Olympus FV3000, Japan).

2.9 Fluorescence in situ hybridization (FISH)

To detect the subcellular location of 4930544M13Rik-201, 18s (positive control), and U6 (negative control) in the TG, a series of Cy3-labeled probes (Table S4) were synthesized by Genepharma (Shanghai, China). FISH was conducted using the FISH kit for frozen sections (Genepharma, China) following the manufacturer's instructions. To investigate the co-expression of 4930544M13Rik-201 and specific proteins, the combined FISH and IF (FISH-IF) were carried out based on previous studies with minor modifications [27]. In brief, prepared sections were desiccation at room temperature for 30 minutes, then rinsed with 1X SSC twice. After antigen retrieval, the sections were washed and dehydrated in a series of ethanol (50, 70, 90, and 100%, 3 min each) and air-dried for 10 min. Pre-warmed prehybridization buffer was gently dispensed onto individual slides, allowing them to prehybridize for 20 minutes within a humid chamber at 47°C. Subsequently, the probes were diluted with a hybridization buffer and subjected to denaturation at 65°C for 10 min, followed by immediate placement on ice. Post prehybridization buffer removal, 100 µl aliquots of each probe were carefully applied onto distinct slides. Then all slides underwent an overnight hybridization for 16 h at 37℃ in a light-protected environment. After hybridization, a sequential series of washes was conducted: 4× SSC (37℃, 5 min×2), 2× SSC (37℃, 5 min), 1× SSC (37℃, 5 min), 0.1× SSC (37℃, 5 min) and followed by a 5-minute rinse with 1X PBS at room temperature. Subsequently, the slides were subjected to IF staining as mentioned in the previous section.

2.10 Statistical analysis

Data analysis was performed by SPSS software (IBM, Chicago), and the results were depicted as the mean accompanied by the standard deviation (SD). The time trend of behavioral data was assessed using a repeated-measures analysis of variance (ANOVA) followed by the Least significant difference (LSD) test. In addition, one-way ANOVA followed by the LSD test was applied to disclose the differences within three or more groups. And two-way ANOVA followed by the LSD was used to determine statistical differences within three groups of certain time points of behavioral data. An unpaired t-test was used to determine statistical differences between the baseline and certain time points of behavioral data and between two groups of PCR and WB data. Statistical significance was determined at a threshold of P < 0.05.

3.1 Up-regulated lncRNA 4930544M13Rik-201 in the TG contributes to trigeminal NP following CCI-ION

Based on prior RNA sequencing analyses, it was observed that the lncRNA 4930544M13Rik-201 exhibited a distinctive upregulation within the TG [13] and DRG [14,15] of three different NP models initiated by peripheral nerve injuries (Fig. 1A, B). To further investigate the expression pattern and role of 4930544M13Rik-201, the trigeminal NP mouse model was established through CCI-ION, which suffered from facial allodynia on days 1 to 28 after surgery (Fig. S1). The results showed that 4930544M13Rik-201 in the TG of the CCI-ION group was significantly upregulated on postoperative days 4, 7, and 14 compared to the Sham group (Fig. 1C). Moreover, the significant increase of 4930544M13Rik-201 in the TG was also observed in female mice following CCI-ION surgery compared with Sham group (Fig. 1D). Besides the lighting 4930544M13Rik-201 staining of cytoplasm of TG neurons, the combined FISH and IF staining of TG sections demonstrated that 4930544M13Rik-201 was majorly stained in the nuclei of TG neurons (Fig. 1E), indicating 4930544M13Rik-201 was mainly localized in the nucleus of TG neurons.

The effects of 4930544M13Rik-201 on trigeminal NP were investigated through knockdown and overexpression of 4930544M13Rik-201 in the TG of mice. The chemically modified siRNA targeted 4930544M13Rik-201 depressed the 4930544M13Rik-201 level two days after injection compared to the negative control group (Fig. 1F). Then 4930544M13Rik-201 were overexpressed in the TG by overexpression plasmid (Fig. 1G). The ipsilateral HWT in CCI-ION mice transfected with siRNA targeted 4930544M13Rik-201 was elevated on postoperative day 7 (Fig. 1H), while a significant decrease in HWT was observed in WT mice on days 1, 2, and 3 postinjection (Fig. 1I). These findings suggested that 4930544M13Rik-201 contribute to the development of trigeminal NP.

3.2 CACNA2D1 regulated by lncRNA 4930544M13Rik-201 in the TG promotes trigeminal NP

To investigate the molecular mechanisms underlying the effects of 4930544M13Rik-201 on trigeminal NP, a co-expression analysis was conducted between 4930544M13Rik-201 and pain-related differential expressed genes (DEGs) [28,29] according to our previous RNA-seq datasets [13] (Fig. 2A). Ten pain-related DEGs was predicted to be highly associated with 4930544M13Rik-201, including Cacna2d1 and Gal, which have been proved to be involved in trigeminal NP in our previous research [30,26]. To further investigate whether Cacna2d1 and Gal were regulated by 4930544M13Rik-201, the expression of 4930544M13Rik-201 in the TG of male mice was silenced or enhanced by siRNA or pcDNA3.1-lnc, respectively. The results demonstrated that the expression of Cacna2d1 mRNA was significantly decreased or increased by both knockdown or overexpression of 4930544M13Rik-201, respectively. While the expression of Gal was not significantly influenced (Fig. 2B, C). Indeed, the expression of mRNA and protein levels were increased in the TG of CCI-ION mice compared to the Sham group 7 days after surgery (Fig. 2D, E). Moreover, the expression of CACNA2D1 protein could be suppressed by knockdown of 4930544M13Rik-201 in the TG of CCI-ION mice (Fig. 2F). Similarly, CACNA2D1 was elevated following overexpression of 4930544M13Rik-201 in the TG of WT mice (Fig. 2G). Furthermore, CACNA2D1 was identified to co-localize with 4930544M13Rik-201 in the TG neurons of CCI-ION mice (Fig. 2H). Finally, inhibiting the activation of CACNA2D1 through intra-ganglionic injection of GBP (inhibitor of CACNA2D1) alleviated the mechanical sensitivity induced by CCI-ION (Fig. I) or overexpression of 4930544M13Rik-201 in the TG (Fig. J). These findings suggest that CACNA2D1 is the downstream target of lncRNA 4930544M13Rik-201 and plays a crucial role in the pathological process of trigeminal NP following trigeminal nerve injury.

3.3 hnRNPA2B1 interacts with both lncRNA 4930544M13Rik-201 and Cacna2d1 mRNA

The above experiments revealed that lncRNA 4930544M13Rik-201 can regulate the expression of CACNA2D1, however, the underlying mechanism is unknown. Nuclear-localized lncRNAs can interact with RNA-binding proteins to influence the expression of target genes [31], therefore, RNA pull-down and LC-MS/MS were carried out to identify the 4930544M13Rik-201-binding proteins in the TG of CCI-ION mice by a pair of sense and antisense probes of 4930544M13Rik-201. In total, 821 proteins were detected to bind with 4930544M13Rik-201 (Table S5). Subsequent GO enrichment analysis was performed on the detected 821 proteins to explore the potential RNA-binding proteins. Among the top 20 enriched proteins in RNA-binding term (Fig. 3A), hnRNPA2B1 (a heterogeneous nuclear ribonucleoprotein) has been shown to regulate gene expression at the post-transcriptional level via interacting with lncRNAs [31,32]. Besides, the results of RNA pull-down and WB further demonstrated the sense probe of 4930544M13Rik-201 bound with hnRNPA2B1 protein (Fig. 3B). Although, WB results indicated that trigeminal nerve injury did not affect the expression of hnRNPA2B1 in the TG (Fig. 3C). Notably, the RIP assay demonstrated that hnRNPA2B1 is not only specifically bound to 4930544M13Rik-201 but also Cacna2d1 mRNA in the TG of CCI-ION mice (Fig. 3D, E). Moreover, the FISH and IF staining of TG sections demonstrated that hnRNPA2B1 was not only co-localized with 4930544M13Rik-201 in the nuclei of TG neurons but also co-localized in the TG neurons expressing CACNA2D1 (Fig. 3F). These findings indicate that hnRNPA2B1 can interact with both 4930544M13Rik-201 and Cacna2d1 mRNA in the nuclei of TG neurons, which may play a crucial role in the regulatory effect of 4930544M13Rik-201 on Cacna2d1 mRNA expression in the TG neurons.

3.4 LncRNA 4930544M13Rik-201 regulates the expression of Cacna2d1 mRNA via hnRNPA2B1

To further confirm the role of hnRNPA2B1 in the regulatory relationship between 4930544M13Rik-201 and Cacna2d1 mRNA, hnRNPA2B1 siRNA was injected in the TG of CCI-ION and 4930544M13Rik-201-overexpressed mice. The downregulation of Cacna2d1 mRNA and CACNA2D1 was observed after hnRNPA2B1 knockdown compared to the groups injected with siRNA-NC (negative control) both in the CCI-ION and 4930544M13Rik-201-overexpressed mice (Fig. 4A-D). In addition, a significant reduction in the expression of 4930544M13Rik-201 was also observed in CCI-ION mice after administration of hnRNPA2B1 siRNA (Fig. 5E), while knockdown of 4930544M13Rik-201 did not significantly affect the expression level of hnRNPA2B1 (Fig. 5F). These results further supported the notion that hnRNPA2B1 was involved in the regulation of Cacna2d1 mRNA expression by 4930544M13Rik-201 in the TG following trigeminal nerve injury.

3.5 Knockdown of hnRNPA2B1 attenuated facial allodynia evoked by CCI-ION and overexpression of lncRNA 4930544M13Rik-201

To identify the effect of hnRNPA2B1 on the trigeminal NP, hnRNPA2B1 siRNA was injected in the TG of CCI-ION and 4930544M13Rik-201-overexpressed mice to successfully silence the expression of hnRNPA2B1 (Fig. 5A). Behavioral tests demonstrated that hnRNPA2B1 siRNA administration significantly reversed the reduction of HWT of CCI-ION or 4930544M13Rik-201-overexpressed mice compared to Sham or negative control group, respectively (Fig. 5B, C). These findings indicate that hnRNPA2B1 is an obligatory effector in the process of the development of trigeminal NP after trigeminal nerve injury.

Recent studies have shown that abnormal expression of lncRNAs was significantly associated with NP [33,34], which could be potential therapeutic targets for NP [12]. To identify a potential lncRNA regulating the development of NP both in the trigeminal and spinal peripheral nervous system, our previous transcriptome sequencing results of TG and the other two previous transcriptome sequencing results of DRG were reanalyzed [13–15]. It has been found that lncRNA 4930544M13Rik-201 was significantly upregulated in both TG and DRG following peripheral nerve injury [13–15], indicating 4930544M13Rik-201 might play an important role in trigeminal and spinal nervous paresthesia. To further identify the role and regulatory mechanisms of 4930544M13Rik-201 following nerve injury, in this study, we confirmed the prolonged upregulation of 4930544M13Rik-201 in the TG of both male and female CCI-ION mice, which is a classical trigeminal NP model [22,25]. FISH and IF demonstrated that 4930544M13Rik-201 was mainly nuclear localized in the TG neurons. Further gain-of-function and loss-of-function studies revealed that 4930544M13Rik-201 participated in the initiation and maintenance of CCI-ION-induced orofacial mechanical allodynia through regulating the expression of CACNA2D1, in which process hnRNPA2B1 played an important role through interacting with 4930544M13Rik-201 and Cacna2d1 mRNA. Moreover, the knockdown of hnRNPA2B1 also attenuated facial allodynia evoked by CCI-ION and overexpression of lncRNA 4930544M13Rik-201. These results suggest that lncRNA 4930544M13Rik-201 could serve as a diagnostic marker for NP responding to peripheral nerve injury. Additionally, it might function as a key potential regulator in the development of NP in conjunction with CACNA2D1 and hnRNPA2B1.

LncRNAs usually cannot code proteins, however, they can regulate the expression of proteins to play a crucial role in the biological processes through interactions with RNA binding proteins (RBPs), RNA, and DNA [35]. In the present study, based on a co-expression analysis conducted between 4930544M13Rik-201 and pain-related differential expressed genes (DEGs), Cacna2d1 mRNA was determined as a potential target of 4930544M13Rik-201. Notably, the expression of Cacna2d1 mRNA and CACAN2D1 protein were significantly decreased or increased after both knockdown or overexpression of 4930544M13Rik-201 in the TG of mice, respectively. Therefore, Cacan2d1 mRNA was determined as a downstream target of lncRNA 4930544M13Rik-201 under the condition of peripheral nerve injury.

Besides the well-known competitive endogenous RNA (ceRNA) mechanism (lncRNA-miRNA-mRNA networks), which majorly takes place in the cytoplasm, lncRNA-RBP networks have gained increasing attention in their role in different diseases, which takes place in the cell nucleus [8,36]. For example, lncRNA UC.25 interacts with signal transducers and activators of transcription1 (STAT1) to regulate the expression of the P2Y₁₄ receptor in spinal microglia, promoting diabetic neuropathic pain [37]. In this study, FISH and IF staining demonstrated that lncRNA 4930544M13Rik-201 was expressed majorly in the nucleus of TG neurons. Interestingly, lncRNAs expressed in the nucleus can regulate target genes by affecting cellular biological processes such as alternative splicing, chromatin status, epigenetic regulation, stability and nucleocytoplasmic transport of mRNAs via interacting with RBPs, transcription factors, export factors and so on [36,38]. To unravel the regulatory mechanism between 4930544M13Rik-201 and Cacna2d1mRNA, this study employed RNA pull-down combined with LC-MS/MS or WB and RIP to identify that RBP hnRNPA2B1 could bind with both 4930544M13Rik-201 and Cacna2d1 mRNA. Furthermore, FISH and IF showed that 4930544M13Rik-201 and hnRNPA2B1 were co-expressed in the nuclear of TG neurons, which also expressed CACNA2D1 in the cytoplasm and membrane. These results support that hnRNPA2B1 interacts with both 4930544M13Rik-201 and Cacan2d1 mRNA in the nuclear of TG neurons, which might account for the regulatory effect of 4930544M13Rik-201 on the Cacan2d1 mRNA.

HnRNPA2B1 is an RBP that belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family [39]. It has diverse functions in RNA metabolism, including pre-mRNA splicing, mRNA stability, transport, and translation regulation [39]. Recent study has indicated that hnRNPA2B1 can serve as an m⁶A reader to stabilize lncRNAs [40]. Briefly, hnRNPA2B1 interacted with and stabilized lncRNA NEAT1 in an m⁶A-dependent manner, which contributed to the maintenance of stemness property via Wnt/β-catenin pathway and exacerbate chemoresistance in gastric carcinoma [40]. Interestingly, the increased expression of 4930544M13Rik-201 in CCI-ION mice was also countered by the administration of hnRNPA2B1 siRNAs in the TG, but total hnRNPA2B1 was not affected by knocking down 4930544M13Rik-201 or CCI-ION treatment. Therefore, in the present study, hnRNPA2B1 might also stabilize 4930544M13Rik-201 through binding to the m⁶A site of 4930544M13Rik-201. However, further studies are needed to investigate whether 4930544M13Rik-201 were methylation modificated following nerve injury and hnRNPA2B1 could directly bind to the m⁶A motif of 4930544M13Rik-201.

Remarkably, besides the interaction with 4930544M13Rik-201, hnRNPA2B1 was also proved to bind with Cacna2d1 mRNA in the TG by RIP. In this study, upregulation of Cacna2d1 mRNA and CACNA2D1 in the TG of CCI-ION mice was suppressed by knockdown of hnRNPA2B1, and knockdown of hnRNPA2B1 also alleviated orofacial mechanical allodynia of CCI-ION and 4930544M13Rik-201-overexpressed mice, similar to the effect of silencing of 4930544M13Rik-201 in the TG of CCI-ION mice. Moreover, It is well known that hnRNPA2B1 can bind to mRNAs and strengthen their stability [39]. Additionally, previous studies have reported that dysregulation of the relationship between lncRNAs and hnRNPA2B1 has been implicated in various diseases, including cancers [41–43] and neurodegenerative disorders [44–46]. Thus, hnRNPA2B1 and 4930544M13Rik-201 might form a complex in the regulation of Cacna2d1 mRNA stabilization. Moreover, as an m⁶A reader, hnRNPA2B1 selectively mediates m⁶A-tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex [38], which implys that hnRNPA2B1—4930544M13Rik-201 complex might not only promote the stability of Cacna2d1mRNA but also nuclear export of Cacna2d1mRNA in the neurons following peripheral nerve injury, contributing to the expression of CACNA2D1 protein. However, further studies are required to investigate the precise molecular mechanism of Cacna2d1 expression regulated by the 4930544M13Rik-201-hnRNPA2B1 complex in the neurons.

CACNA2D1, known colloquially as a voltage-activated calcium channel (VGCC) subunit, is a newly discovered NMDAR-interacting protein involved in the development of NP [47]. Our previous study has also demonstrated that CACNA2D1 interacts with NMDAR1 in the TG neurons, contributing to the orofacial ectopic pain induced by trigeminal nerve transection injury [26]. Similarly, in the present study, increased expression of Cacna2d1 mRNA and CACNA2D1 was detected in the TG in CCI-ION mice, and the HWT was reversed by administration of CACNA2D1 inhibitory ligands in a dose-dependent manner, which suggested that CACNA2D1 in the TG neurons promoted the development of trigeminal NP following trigeminal nerve injury. Moreover, studies have shown that increased CACNA2D1 expression contributes to enhanced calcium influx into neurons, amplifying pain signaling in the central nervous system [17,18]. This study revealed that 4930544M13Rik-201 was co-localized with CACNA2D1 in the same neurons in the TG, and the expression of Cacna2d1 and CACNA2D1 was positively regulated by 4930544M13Rik-201, while inhibition of CACNA2D1 suppressed the decrease HWT in 4930544M13Rik-201 overexpressed mice. These findings suggest that CACNA2D1, as a downstream target of 4930544M13Rik-201 was involved in the regulation of NP following peripheral nerve injury.

This study identified a significant increase in lncRNA 4930544M13Rik-201 in the TG neurons of mice following trigeminal nerve injury, which contributed to the development of trigeminal NP (Fig. 6). Moreover, 4930544M13Rik-201 interacted with hnRNPA2B1, promoting the stabilization of Cacna2d1 mRNA and upregulating CACNA2D1 expression. Inhibition of CACNA2D1 and silencing of hnRNPA2B1 both alleviated allodynia induced by CCI-ION and overexpression of 4930544M13Rik-201 in the TG. These findings emphasize the role of 4930544M13Rik-201 in NP and underscore its potential as a biomarker for pain diagnosis. Additionally, these results suggest a new therapeutic strategy for pain management by targeting the 4930544M13Rik-201—hnRNPA2B1—CACNA2D1 axis.

Supplementary Information Supplementary materials will be available in the online version.

Acknowledgments We thank Ms. Xin Guan and Mr. Fan-Zi Wu for their technical assistance.

Author Contribution The experiments and data collection were executed by Zhonghan Fang and Fei Liu. The analysis of data was conducted by Qingfeng Tang and Yajing Liu. Data curation and investigation were conducted by Yuheng Feng, Minghan Ye, and Zhongyuan Wu. Data interpretation was provided by Yuan Peng, Zheng Zheng, and Qijing Li. The figures were prepared by Zhonghan Fang, Fei Liu, and Yanyan Zhang. Jiefei Shen agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Jiefei Shen, Hang Wang, and Fei Liu provided financial support and study design. The first draft of the manuscript was written by Zhonghan Fang and Fei Liu. All authors review and edit the previous version of the manuscript. All the authors have read and approved the final manuscript.

Funding This study was funded by the National Natural Science Foundation of China (Grant No. 82071149, 82271014), the Department of Science and Technology of Sichuan Province (Grant No. 2024NSFSC0683, 2024NSFSC1594).

Data Availability The data supporting the findings of this study can be obtained from the corresponding author upon reasonable request.

Declarations

Ethics Approval Animal subjects for this study were utilized in compliance with ethical guidelines outlined by the Animal Care and Use Committee at Sichuan University Health Science, all experimental procedures involving animals were approved by the Committee of West China Hospital of Stomatology Sichuan University (NO: WCHSIRB-D-2021-56).

Consent to Participate N/A.

Consent for Publication N/A.

Conflict of Interest The authors declare that there is no conflict of interest.

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LncRNA 4930544M13Rik-201 regulates CACNA2D1 expression via interacting with hnRNPA2B1 to promote neuropathic pain following nerve injury

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Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Animals and neuropathic pain model

2.2 Behavioral test

2.3 Administration of drugs into the TG

2.4 Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

2.5 Western blotting (WB)

2.6 RNA pull-down

2.7 RNA immunoprecipitation (RIP)

2.8 Immunofluorescence (IF)

2.9 Fluorescence in situ hybridization (FISH)

2.10 Statistical analysis

3. Results

3.1 Up-regulated lncRNA 4930544M13Rik-201 in the TG contributes to trigeminal NP following CCI-ION

3.2 CACNA2D1 regulated by lncRNA 4930544M13Rik-201 in the TG promotes trigeminal NP

3.3 hnRNPA2B1 interacts with both lncRNA 4930544M13Rik-201 and Cacna2d1 mRNA

3.4 LncRNA 4930544M13Rik-201 regulates the expression of Cacna2d1 mRNA via hnRNPA2B1

3.5 Knockdown of hnRNPA2B1 attenuated facial allodynia evoked by CCI-ION and overexpression of lncRNA 4930544M13Rik-201

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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