Retracted Article: Gm5820, an antisense RNA of FGF1, suppresses FGF1 expression at the posttranscriptional level to inactivate the ERK/STAT3 pathway and alleviates neuropathic pain in mice

Abstract

Emerging evidence reveals that lncRNAs play important roles in various pathological processes, but precious little indicates their regulatory role in neuropathic pain. In this study, we performed unbiased whole transcriptome profiling in dorsal root ganglions (DRGs) from mice with sham operation and mice with chronic constriction injury (CCI). Gm5820 was one of the most downregulated RNA transcripts in CCI neuropathic pain model mice. Then, a pcDNA-Gm5820 expression vector was constructed and administered into CCI mice through intrathecal injection. The results showed that upregulation of Gm5820 alleviated mouse mechanical allodynia and thermal/cold hyperalgesia, and reduced the accumulation of inflammatory cytokines and ROS in the DRG tissue. Moreover, different concentrations of pcDNA-Gm5820 expression vector and Gm5820 siRNA were respectively transfected into primary DRG neurons from 4^th to 6^th lumbar vertebra (L4-L6). We found that Gm5820 overexpression improved cell viability and migration, and reduced the production of ROS, LDH and IL-1β. In contrast, Gm5820 knockdown had the opposite effects. Furthermore, RNA pull-down assays with FGF1 and Gm5820 cDNA probes both demonstrated that FGF1 mRNA and Gm5820 directly bound to each other. Moreover, Gm5820 negatively regulated the stability of FGF1 mRNA. Gm5820 suppressed the expression of FGF1 at the post-transcriptional level and negatively regulated the activation of ERK1/2-mediated STAT3, a critical contributor in neuropathic pain. In conclusion, Gm5820 directly binds to FGF1 mRNA and suppresses FGF1 expression at the posttranscriptional level, it functions as a negative regulator in the activation of the ERK/STAT3 pathway, and upregulation of Gm5820 alleviates neuropathic pain in CCI mice.

Fig. 1. Differentially expressed RNA transcripts induced by CCI and qPCR confirmation for their expression. (A) Length distribution of differentially expressed RNA transcripts induced by CCI. (B) Chromosomal distribution of differentially expressed RNA transcripts induced by CCI. (C) The expression of the top 10 up-regulated RNA transcripts were confirmed by qPCR. (D) The expression of the top 10 down-regulated RNA transcripts were confirmed by qPCR. CCI model was established with the ligation of sciatic nerves. DRG tissues were respectively obtained from L4–L6 of 6 sham and 6 CCI mice at 72 h post the operation. Total RNA was isolated, strand-specific barcoded libraries were generated, and RNA sequencing was performed on the HiSeq 2500. Transcripts were annotated using publicly available datasets from RefSeq for protein-coding transcripts and the Human Body Map lncRNAs annotation for long noncoding genes. RNA transcripts with a fold change of 2.0 and a false discovery rate-adjusted P value of <0.05 were considered as differentially expressed. A total of 1322 differentially expressed transcripts were found and summarized. The expression of the top 10 up-regulated and 10 down-regulated RNA transcripts were confirmed by qPCR. N = 4, *P < 0.05 compared with sham.

Fig. 2. Gm5820 was significantly downregulated during development of neuropathic pain in CCI mice. (A) The withdrawal to mechanical latencies stimulus of the sham and CCI mice from day 0 to day 21 post CCI surgery. (B) The withdrawal latencies to thermal stimulus of the sham and CCI mice from day 0 to day 21 post CCI surgery. (C) The withdrawal latencies to cold stimulus of the sham and CCI mice from day 0 to day 21 post CCI surgery. (D) Gm5820 expression was significantly downregulated during development of neuropathic pain in CCI mice. The time course of difference scores in the withdrawal latencies of the sham and CCI mice to mechanical, thermal, and cold stimuli were tested at days 0, 3, 7, 14 and 21 post CCI surgery. The levels of Gm5820 RNA in DRG tissues of the sham and CCI mice were detected with qPCR. N = 6, *P < 0.05, **P < 0.01 compared with sham.

Fig. 3. Intrathecal overexpression of Gm5820 alleviated neuropathic pain in CCI mice. (A) Intrathecal injection of pcDNA-Gm5820 distinctly increased the expression of Gm5820 RNA in DRG neurons of CCI mice. (B) Intrathecal overexpression of Gm5820 decreased the withdrawal latency of CCI mice to mechanical stimulus. (C) Intrathecal overexpression of Gm5820 decreased the withdrawal latency of CCI mice to thermal stimulus. (D) Intrathecal overexpression of Gm5820 decreased the withdrawal frequency of CCI mice to cold stimulus. (E) Intrathecal overexpression of Gm5820 sharply reduced the expression of IL-1β and TNF-α proteins in DRG neurons of CCI mice. (F) Intrathecal overexpression of Gm5820 sharply reduced the accumulation of ROS in DRG neurons of CCI mice. A total of 45 male C57BL/6 mice were randomly divided into 3 groups, including 15 mice with sham operation (sham), 15 CCI mice administered with 5 mg kg⁻¹ empty vector by intrathecal injection (CCI-vector), and 15 CCI mice administered with 5 mg kg⁻¹ pcDNA-Gm5820 by intrathecal injection (CCI-Gm5820). The levels of Gm5820 RNA in DRG neurons of each group were detected with qPCR. The time course of difference scores in the withdrawal latencies of each group to mechanical, thermal, and cold stimuli were tested at days 0, 3, 7 and 14 post CCI surgery. Protein levels of IL-1β and TNF-α in the DRG neurons of each group were detected with western blotting. ROS contents in the spinal cord neurons of each group were detected with DCFH-DA ROS Assay kit. N = 15, *P < 0.05 compared with sham, ^#P < 0.05 compared with CCI-vector.

Fig. 4. Gm5820 overexpression increased cell proliferation and migration and reduced oxidative stress and inflammation in mouse primary DRG neurons. (A) The overexpression efficiencies of different concentrations of pcDNA-Gm5820 in mouse primary DRG neurons. (B) Gm5820 overexpression increased cell proliferation of mouse primary spinal cord neurons in a dose-dependent manner. (C) Gm5820 overexpression increased cell migration of mouse primary DRG neurons in a dose-dependent manner. (D) Gm5820 overexpression decreased ROS accumulation in mouse primary DRG neurons in a dose-dependent manner. (E) Gm5820 overexpression decreased LDH release in mouse primary DRG neurons in a dose-dependent manner. (F) Gm5820 overexpression decreased IL-1β secretion in mouse primary DRG neurons in a dose-dependent manner. Primary DRG neurons were isolated from L4–L6 of 3 months old mouse and cultured in vitro. The pcDNA-Gm5820 at the concentrations of 0.1, 0.5 and 1 μg mL⁻¹ were respectively transfected into the cells. At 72 h post transfection, the cells of each group were harvested. CCK-8 assay was used to detect cell proliferation, and Transwell Cell Migration assay was used to detect cell migration. ROS accumulation in the cells were detected with DCFH-DA ROS Assay kit. LDH and II-1β contents in the supernatant were detected with LDH Detection Kit and IL-1β ELISA Kit. N = 5, *P < 0.05, **P < 0.01 compared with vector.

Fig. 5. Gm5820 knockdown inhibited cell proliferation and migration and promoted oxidative stress and inflammation in mouse primary DRG neurons. (A) The knockdown efficiencies of different concentrations of Gm5820 siRNA in mouse primary DRG neurons. (B) Gm5820 knockdown decreased cell proliferation of mouse primary DRG neurons in a dose-dependent manner. (C) Gm5820 knockdown decreased cell migration of mouse primary DRG neurons in a dose-dependent manner. (D) Gm5820 knockdown enhanced ROS accumulation in mouse primary DRG neurons in a dose-dependent manner. (E) Gm5820 knockdown enhanced LDH release in mouse primary DRG neurons in a dose-dependent manner. (F) Gm5820 knockdown enhanced IL-1β secretion in mouse primary DRG neurons in a dose-dependent manner. Primary DRG neurons were isolated from L4–L6 of 3 months old mouse and cultured in vitro. The Gm5820 siRNA at the concentrations of 20 and 40 nM were respectively transfected into the cells. At 72 h post transfection, the cells of each group were harvested. CCK-8 assay was used to detect cell proliferation, and Transwell Cell Migration assay was used to detect cell migration. ROS accumulation in the cells was detected with DCFH-DA ROS Assay kit. LDH and II-1β contents in the supernatant were detected with LDH Detection Kit and IL-1β ELISA Kit. N = 5, *P < 0.05, **P < 0.01 compared with scramble.

Fig. 6. Gm5820 directly bound to FGF1 mRNA and suppressed FGF1 expression at the posttranscriptional level. (A) The landscape of Gm5820 gene in the genome reveals that Gm5820 was in the antisense strand of FGF1. (B) Directly binding of Gm5820 RNA and FGF1 mRNA verified by RNA pull-down assay with biotinylated cDNA probe of FGF1 mRNA. (C) Directly binding of Gm5820 RNA and FGF1 mRNA verified by RNA pull-down assay with biotinylated cDNA probe of Gm5820 RNA. Primary DRG neurons were isolated from L4–L6 of 3 months old mouse and cultured in vitro. Biotinylated cDNA probes of FGF1 mRNA and Gm5820 RNA were applied into RNA pull-down assays. Then, northern blotting and qPCR were respectively used to detect the expression of Gm5820 RNA and FGF1 mRNA in the pulled down RNA complex. ***P < 0.001. (D) The regulation of Gm5820 on expression of FGF1 mRNA. (E) The regulation of Gm5820 on expression of FGF1 protein. 0.5 μg mL⁻¹ pcDNA empty vector, 0.5 μg mL⁻¹ pcDNA-Gm5820, 40 nM scrambled siRNA, 40 nM Gm5820 siRNA and 40 nM scrambled siRNA plus 1 nM SCH772984 (Selleck Chemicals, Houston, TX) were treated with primary DRG neurons. At 72 h post treatment, expression of Gm5820 RNA and the mRNA levels of FGF1, ERK1/2 and STAT3 were detected with qPCR, and the protein levels of FGF1, ERK1/2 and STAT3 were detected with western blotting. *P < 0.05.