Mechanism Study of Circular RNA-GRIN2B Inhibiting Neuropathic Pain

Background Introduction

Neuropathic pain is a persistent pain caused by damage to the somatic sensory nervous system, with its etiology not yet fully understood and clinical treatment facing many difficulties. Given the complexity of neuropathic pain, there is an urgent need to reveal its molecular mechanisms to find early intervention strategies and effective drug targets. In previous studies, the sodium-dependent potassium channel Slick (targeting gene sequences similar to medium-conductance potassium channels) was found to play an important role in regulating pain perception and inflammatory responses. Nevertheless, the specific functions of circular RNAs (circRNAs) in neuropathic pain remain unclear.

Studies related to the Slick channel indicate that NF-κB is a key promoter regulating Slick gene transcription. Meanwhile, circRNAs are gradually becoming potential molecular diagnostic markers and therapeutic targets due to their structural stability, abundant expression, tissue specificity, and developmental stage specificity. Therefore, the purpose of this study is to explore the biological function of circGRIN2B in neuropathic pain and its possible molecular mechanisms.

Research Source

This paper was written by researchers Kun Wang, Zicong Shen, Xin Peng, Xiaotao Wu, and Lu Mao, affiliated with the Medical School of Southeast University, Zhongda Hospital Affiliated to Southeast University in Nanjing, China, and the School of Biomedical Engineering and Informatics, Nanjing Medical University. The paper was published in “Neuromolecular Medicine” in 2024.

Research Process

The study subjects included two-month-old male Sprague-Dawley rats weighing 230-250 grams. The chronic constriction injury (CCI) model was established using Xie’s method (1988) to simulate neuropathic pain. The experimental rats were randomly divided into six groups (six rats per group): Control+NC group (saline as negative control), Control+OE-circGRIN2B group (overexpressing circGRIN2B), Control+sh-circGRIN2B group (inhibiting circGRIN2B expression), CCI+NC group, CCI+OE-circGRIN2B group, and CCI+sh-circGRIN2B group. Catheters were successfully implanted using the lumbar puncture method, followed by drug injection. To study the biological function of circGRIN2B, various experiments were designed, including fluorescence in situ hybridization, whole-cell patch-clamp technique, real-time quantitative PCR, Western blot, immunofluorescence, RNA pull-down, mass spectrometry analysis, RNA immunoprecipitation (RIP), enzyme-linked immunosorbent assay (ELISA), etc.

Fluorescence In Situ Hybridization (FISH)

The expression location of circGRIN2B in rat DRG neurons was observed under a fluorescence microscope, showing that calcitonin gene-related peptide (CGRP) was also positively expressed in neurons stained with circGRIN2B.

Whole-Cell Patch-Clamp Technique

Electrophysiological recordings were performed on DRG neurons treated differently (wild-type and circGRIN2B knockout groups). The results showed that after knocking out circGRIN2B, the total potassium ion current (I_K) with a 20mV incremental ladder significantly decreased, indicating that circGRIN2B knockout may promote neuronal excitability.

Real-Time Quantitative PCR and Western Blot

RT-qPCR and Western blot analysis found that only after knocking down circGRIN2B, the Slick channel protein and gene expression were significantly reduced, suggesting that circGRIN2B may affect DRG neuron excitability by regulating Slick levels.

RNA Pull-Down and Mass Spectrometry Analysis

RNA pull-down experiments were performed using biotin-labeled probes. Silver staining and mass spectrometry analysis showed that the protein bound to circGRIN2B was p65, confirming direct interaction between circGRIN2B and the main component of NF-κB, p65.

RNA Immunoprecipitation (RIP) and ELISA

The binding of circGRIN2B to p65 was verified through RIP experiments. ELISA results showed that compared with the CCI+NC group, the protein levels of inflammatory factors (IL-1β, IL-6, TNF-α) were significantly increased in the CCI+sh-circGRIN2B group, while significantly decreased in the CCI+OE-circGRIN2B group, indicating that circGRIN2B may participate in the process of neuropathic pain by mediating the levels of inflammatory cytokines.

Main Results

1. Verified the expression of circGRIN2B in DRG neurons and its translocation from cytoplasm to nucleus under IL-1β stimulation.
2. Found that knockdown of circGRIN2B significantly reduced sodium-activated outward potassium currents, thereby increasing neuronal excitability.
3. Confirmed through RNA pull-down and RIP experiments that circGRIN2B directly interacts with NF-κB p65 and promotes Slick gene expression.
4. CCI rat model studies showed that increasing circGRIN2B expression could significantly alleviate mechanical and thermal hyperalgesia while reducing inflammatory factor levels.
5. NF-κB binds to the Slick promoter in DRG tissues, regulating its expression, indirectly confirming that circGRIN2B regulates Slick through NF-κB.

Conclusion

This study reveals the mechanism by which circGRIN2B inhibits neuropathic pain by interacting with NF-κB p65 to regulate Slick expression. This finding suggests that the circGRIN2B/NF-κB/Slick pathway may be a new therapeutic strategy, and its intervention may open up new avenues for effective treatment of neuropathic pain.

Research Significance

This paper makes a major breakthrough in the molecular mechanism research of neuropathic pain, providing potential targets for new treatment methods. By deeply analyzing the mechanism of circGRIN2B in DRG neurons, it provides important theoretical basis and experimental foundation for future use of circRNA in treating neuropathic pain.

Acknowledgments and Contributions

The authors express gratitude to all personnel involved in design, material preparation, data collection, and analysis, and mention that this study was supported by the National Natural Science Foundation of China (Grant No. 81902252).

This paper has important academic value and clinical significance for understanding the role of circRNA in neuropathic pain, and will help develop more targeted and effective treatment plans for neuropathic pain in the future.