MicroRNA-7a ameliorates neuropathic pain in a rat model of spinal nerve ligation via the neurofilament light polypeptide-dependent signal transducer and activator of transcription signaling pathway

Abstract

Neuropathic pain is a type of chronic pain induced by either central or peripheral nerve injury. MicroRNAs have been recently linked to many diseases, including neuropathic pain. However, the role of miR-7a in neuropathic pain still remains elusive. Thus, we aim to investigate the effects of miR-7a on neuropathic pain based on the spinal nerve ligation rat model. After establishment of spinal nerve ligation rat models, rats were infected with adeno-associated virus-neurofilament light polypeptide, adeno-associated virus-miR-7a or treated with metformin. The paw withdrawal threshold and paw withdrawal latency were assessed afterward, and the expression of miR-7a and neurofilament light polypeptide as well as their interaction was determined. Subsequently, miR-7a was overexpressed or silenced in dorsal root ganglion cells to investigate the role of miR-7a in neuropathic pain. Furthermore, the regulatory effect of neurofilament light polypeptide on neuropathic pain was detected using plasmid overexpressing neurofilament light polypeptide. Spinal nerve ligation rat model exhibited upregulation of neurofilament light polypeptide but downregulation of miR-7a. In addition, neurofilament light polypeptide accumulation or miR-7a inhibition decreased paw withdrawal threshold and paw withdrawal latency. Then, neurofilament light polypeptide accumulation or miR-7a inhibition was observed to increase the phosphorylation level of signal transducer and activator of transcription. miR-7a was found to directly target neurofilament light polypeptide and downregulate neurofilament light polypeptide. In addition, inhibiting the signal transducer and activator of transcription signaling pathway was also revealed to increase paw withdrawal threshold and paw withdrawal latency. Collectively, our study demonstrated that miR-7a ameliorated neuropathic pain via blocking the signal transducer and activator of transcription signaling pathway by repressing neurofilament light polypeptide. These findings, if taken further, can be of important clinical significance in treating patients with neuropathic pain.

Keywords: Neuropathic pain; microRNA-7a; neurofilament light polypeptide; signal transducer and activator of transcription signaling pathway.

Figure 1.

The SNL rats exhibit upregulation of NEFL. (a) mRNA expression of NEFL in rat dorsal spinal cord L4-L5 DRG tissues in the sham-operated or SNL-treated rats on the 3rd, 7th, and 14th day after operation (n = 15, five rats at each time point); (b) protein level of NEFL in rat dorsal spinal cord L4-L5 DRG tissues in the sham-operated or SNL-treated rats on the 14th day after operation; *p < 0.05 versus the sham-operated rats; (c) expression of NEFL, IB4, and NF200 in rat dorsal spinal cord L4-L5 DRG tissues in rats of the SNL model on the 7th and 14th day after operation measured by immunofluorescence; white arrow represents double staining of NEFL with IB4, and white arrow head represents double staining of NEFL with NF200, scale bar = 25 µm; all data were measurement data, expressed as mean ± standard deviation, and analyzed by un-paired t-test; and the experiment was repeated three times independently. NEFL: neurofilament light polypeptide; SNL: spinal nerve ligation; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

Overexpression of NEFL triggers neuropathic pain. (a) The effect of NEFL accumulation on PWT on the day 0, 3, 7, 10, 12, and 14 after operation; (b) the effect of NEFL accumulation on PWL on the day 0, 3, 7, 10, 12, and 14 after operation; *p < 0.05 versus the sham group; ^#p < 0.05 versus the sham + AAV-NC group; ^&p < 0.05 versus the SNL + AAV-NC group; all data were measurement data, expressed as mean ± standard deviation, and analyzed by repeated measures ANOVA, followed by Tukey’s post hoc tests for multiple comparisons (n = 15). NEFL: neurofilament light polypeptide; SNL: spinal nerve ligation; NC: negative control; AAV: adeno-associated virus.

Figure 3.

Inactivation of STAT3 signaling pathway attenuates neuropathic pain induced by NEFL. (a) PWT on the day 0, 3, 7, 10, 12, and 14 after operation after alteration of NEFL and the STAT3 signaling pathway; (b) PWL on the day 0, 3, 7, 10, 12, and 14 after operation after alteration of NEFL and the STAT3 signaling pathway; (c-d) extent of STAT3 phosphorylation and protein levels of STAT3 in dorsal spinal cord L4-L5 DRG tissues on the 14th day after operation, the 7th day after infection with AAV-NEFL, and the 7th day after administrated with metformin; *p < 0.05 versus the SNL + AAV-NC + saline group; ^#p < 0.05 versus the SNL + AAV-NEFL + saline group; all data were measurement data and expressed as mean ± standard deviation; the data in panel a and b were analyzed using repeated measures ANOVA, followed by Tukey’s post hoc tests for multiple comparisons while the data in panel c were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests for multiple comparisons (n = 15). NEFL: neurofilament light polypeptide; STAT3: signal transducer and activator of transcription; SNL: spinal nerve ligation; NC: negative control; AAV: adeno-associated virus; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 4.

NEFL is a target gene of miR-7a. (a) The binding site between miR-7a and NEFL predicted by online prediction software; (b) the binding of miR-7a to NEFL verified by dual luciferase reporter assay; *p < 0.05 versus co-transfection of NC mimic and WT-NEFL; (c) NEFL expression at mRNA level with overexpression or silencing of miR-7a; (d) expression of NEFL at protein level with overexpression or silencing of miR-7a; *p < 0.05 versus the NC-mimic group; ^#p < 0.05 versus the NC-inhibitor group; all data were measurement data and expressed as mean ± standard deviation and analyzed by un-paired t-test; the experiment was repeated three times independently. miR-7a: microRNA-7a; NEFL: neurofilament light polypeptide; NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MUT: mutant type; WT: wild type.

Figure 5.

miR-7a blocks the STAT3 signaling pathway by downregulating NEFL. The total protein level of STAT3 and the extent of STAT3 phosphorylation in DRG cells after transfected with miR-7a mimic or inhibitor or NEFL detected by western blot analysis; *p < 0.05 versus the NC-mimic group; ^#p < 0.05 versus the NC-inhibitor group; ^$p < 0.05 versus the oe-NC group; ^&p < 0.05 versus the oe-NEFL group; all data were measurement data, expressed as mean ± standard deviation, and analyzed by one-way ANOVA, followed by Tukey’s post hoc tests for multiple comparisons; the experiment was repeated three times independently. miR-7a: microRNA-7a; NEFL: neurofilament light polypeptide: STAT3: signal transducer and activator of transcription; NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 6.

miR-7a attenuates neuropathic pain by suppressing NEFL expression and the STAT3 signaling pathway. (a) miR-7a expression in the sham-operated or SNL-treated rats; *p < 0.05 versus the sham group; (b) PWT of sham-operated or SNL-treated rats after the infection of AAV-miR-7a on the day 0, 3, 7, 10, 12, and 14 after operation; (c) PWL level of sham-operated or SNL-treated rats in response to the infection of AAV-miR-7a on the day 0, 3, 7, 10, 12, and 14 after operation; (d) and (e) protein bands and levels of NEFL and STAT3 as well as the extent of STAT3 phosphorylation in SNL-treated rats in response to infection of AAV-miR-7a at the 14th day after operation; *p < 0.05 versus the sham + AAV-NC group; ^#p < 0.05 versus the SNL + AAV-NC group; ^&p < 0.05 versus the sham + AAV-NEFL + AAV-miR-7a group; all data were measurement data and expressed as mean ± standard deviation; the data in panel A were analyzed using t-test, and data in panel B and C were analyzed using repeated measures ANOVA, followed by Tukey’s post hoc tests for multiple comparisons; while the data in panel E were analyzed using ANOVA, followed by Tukey’s post hoc tests for multiple comparisons (n = 15). miR-7a: microRNA-7a; NEFL: neurofilament light polypeptide; STAT3: signal transducer and activator of transcription; SNL: spinal nerve ligation; AAV: adeno-associated virus; NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 7.

Molecular mechanism underlying how miR-7a alleviates neuropathic pain. miR-7a could specifically target NEFL and inhibit NEFL expression to inactivate the STAT3 signaling pathway, thus ameliorating neuropathic pain. NEFL: neurofilament light polypeptide; STAT3: signal transducer and activator of transcription.