Long Noncoding RNA H19 Induces Neuropathic Pain by Upregulating Cyclin-Dependent Kinase 5-Mediated Phosphorylation of cAMP Response Element Binding Protein

Abstract

Objective: Neuropathic pain (NP) is a debilitating condition caused by nervous system injury and chronic diseases. LncRNA H19 is upregulated in many human diseases, including NP. Cyclin-dependent kinase 5 (CDK5) aggressively worsens inflammatory action and nerve damage to cause severe NP. Phosphorylated cAMP response element binding protein (CREB) is detrimental to nerves and promotes NP progression. Herein, aim of our study was to assess the mechanism of lncRNA H19.

Methods: The NP rat model was established using chronic constriction injury (CCI). Paw withdrawal threshold (PWT) tests and paw withdrawal latency (PWL) tests were performed. Then, small interfering (si)RNA against H19 was intrathecally injected into rats to suppress H19 expression. Schwann cells were isolated from NP rats and transfected with siRNA-H19 or a lentivirus (LV)-based vector expressing H19. Inflammatory factors and glial fibrillary acidic protein (GFAP) were detected. Western blot analysis was conducted to detect CDK5/p35 and p-CREB expression. Finally, H19, CDK5 and CREB phosphorylation were tested with the combination of the CDK5 inhibitor roscovitine and transfection of LV-H19 and siRNA-H19. Finally, we investigated the binding relationships between H19 and miR-196a-5p and between miR-196a-5p and CDK5 and detected the mRNA expression of miR-196a-5p and CDK5 in rats with H19 knockdown and in Schwann cells with H19 knockdown.

Results: Highly expressed H19, CDK5/p-35 and p-CREB were observed in NP rats, accompanied by obviously decreased PWT and PWL, upregulated inflammatory factors and GFAP levels, and reduced 5-HT_2A and GABA_B2 expression. siRNA-H19 restored NP-related indexes and downregulated CDK5/p35 and p-CREB phosphorylation. siRNA-H19, together with the CDK5 inhibitor roscovitine, reduced CDK5 and p-CREB expression in Schwann cells isolated from NP rats. Binding sites between H19 and miR-196a-5p and between miR-196a-5p and CDK5 were identified. Silencing H19 upregulated miR-196a-5p expression and downregulated CDK5 levels.

Conclusion: Our study demonstrated that silencing H19 inhibited NP by suppressing CDK5/p35 and p-CREB phosphorylation via the miR-196a-5p/CDK5 axis, which may provide new insight into NP treatment.

Keywords: cAMP response element binding protein phosphorylation; cyclin-dependent kinases 5; long noncoding RNA H19; neuropathic pain; roscovitine.

Figure 1

CCI induces NP in rats. Rat PWT (A) and PWL (B) were both decreased after CCI operation. (C) ELISA showed that TNF-α, IL-1β and IL-6 levels in spinal dorsal horns of rats were significantly upregulated after CCI operation. (D) Western blot analysis showed that in spinal dorsal horns of CCI rats, GFAP expression increased while 5-HT_2A and GABA_B2 expression decreased. The naive baseline was the value measured on the previous day of the CCI operation. CCI rats were euthanized 24 days after surgery, and the relative indexes were measured. Data are expressed as the mean ± standard deviation. n = 6 in each group. Two-way ANOVA was applied to analyze the data in panels (A and B), and the t-test was used for analyzing comparisons in panels (C and D); compared with the sham group, **p < 0.01. Abbreviations: CCI, chronic constriction injury; NP, neuropathic pain; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; TNF, tumor necrosis factor; IL-1β, interleukin-1β; GFAP, glial fibrillary acidic protein; ANOVA, analysis of variance.

Figure 2

NP rats have increased CREB phosphorylation via the CDK5/p35 axis. (A) H19 expression was clearly promoted as detected by RT-qPCR. (B) Western blot analysis showed that expression of both p35 and CDK5 was enhanced in NP rats. (C) CDK5 expression in the spinal cord dorsal horn was enhanced as assessed by immunofluorescence assay. (D) Western blot analysis showed that expression of both p-CaMK II and p-CREB was enhanced in NP rats. (E) The colocalization of p-CAMKII and p-CREB in spinal dorsal horn neurons (NeuN) was detected by double immunofluorescence. Data are expressed as the mean ± standard deviation, and n = 6 in each group. The t-test was used to analyze comparisons between two groups; compared with the sham group,*p<0.05, **p < 0.01. Abbreviations: CDK5, cyclin-dependent kinase 5; CREB, cAMP response element binding protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NP, neuropathic pain.

Figure 3

siRNA-H19 attenuates NP. (A) H19 expression decreased in NP rats treated with siRNA-H19. siRNA-H19 enhanced (B) PWT and (C) PWL of NP rats. (D) HE staining performed to evaluate the effects of intrathecally installed tubes and transfection reagents on rat spinal dorsal horns showed no evident changes. (E) siRNA-H19 reduced TNF-α, IL-1β and IL-6 expression in rat spinal dorsal horns. (F) Western blot analysis showed that in rat spinal dorsal horns, GFAP expression was downregulated, while 5-HT2A and GABAB2 expression was upregulated. Data are expressed as the mean ± standard deviation, and n = 6 in each group. Two-way ANOVA was applied to analyze data in panels (B and C), and the t-test was used to analyze comparisons in panels (E and F); compared with the NP + siRNA-NC group, **p < 0.01. Abbreviations: si, small interfering; NP, neuropathic pain; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; HE, hematoxylin-eosin; TNF, tumor necrosis factor; IL-1β, interleukin-1β; GFAP, glial fibrillary acidic protein; ANOVA, analysis of variance.

Figure 4

siRNA-H19 downregulates CDK5/p35 and inhibits CREB phosphorylation. (A) Western blot analysis showed that p35 and CDK5 levels in NP rats were decreased by siRNA-H19. (B) Immunofluorescence assay suggested that CDK5 expression in the spinal cord dorsal horn was decreased. (C) Western blot analysis indicated decreased expression of p-CaMKII and p-CREB in NP rats with siRNA-H19. Data are expressed as the mean ± standard deviation, and n = 6 in each group. The t-test was used to analyze comparisons between two groups; compared with the NP + siRNA-NC group, *p < 0.05, **p < 0.01. Abbreviations: si, small interfering; CDK5, cyclin-dependent kinases 5; CREB, cAMP response element binding protein; NP, neuropathic pain.

Figure 5

Effects of siRNA-H19 or LV-H19 and the CDK5 inhibitor roscovitine to reduce Schwann cells, CDK5 and p-CREB. (A) S100 staining confirmed that the isolated cells were Schwann cells. (B) RT-qPCR indicated increased H19 expression in Schwann cells. (C) Western blot analysis showed that GFAP expression in Schwann cells dropped significantly. (D) ELISA showed a decrease in TNF-α, IL-1β and IL-6 expression. (E) Western blot analysis revealed a reduction in CDK5 and p-CREB expression in Schwann cells. The experiments were performed 3 times; data are expressed as the mean ± standard deviation. Two-way ANOVA and Tukey’s multiple comparisons test were applied to determine statistical significance. *p < 0.05, **p < 0.01. Abbreviations: si, small interfering; LV, lentivirus; CDK5, cyclin-dependent kinases 5; CREB, cAMP response element binding protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GFAP, glial fibrillary acidic protein; TNF, tumor necrosis factor; IL-1β, interleukin-1β; ANOVA, analysis of variance.

Figure 6

H19 competitively binds to miR-196a-5p to upregulate cdk5 expression. (A) Bioinformatics analysis and dual-luciferase assay of the H19 and miR-196a-5p binding relationship. (B) The target binding relationship between miR-196a-5p and CDK5 was detected by bioinformatics analysis and dual-luciferase assay. (C) The levels of miR-196a-5p and cdk5 in NP rats were detected by RT-qPCR. (D) RT-qPCR was used to detect the levels of miR-196a-5p and cdk5 in Schwann cells isolated from NP rats. The cell experiment was repeated three times. The sample size of the experiment in vivo was n = 6 for each group. The results are expressed as the mean ± standard deviation. Data in panels (A and B) were analyzed by two-way ANOVA, and data in panels (C and D) were analyzed by one-way ANOVA. **p < 0.01.