LncRNA FTX Involves in the Nogo-66-Induced Inhibition of Neurite Outgrowth Through Regulating PDK1/PKB/GSK-3β Pathway

Abstract

Nogo-66 can inhibit neurite outgrowth, while its regulation mechanisms have not been fully elucidated. Recent studies prove that lncRNAs are involved in neurite outgrowth. This study was aimed to investigate whether lncRNA FTX was involved in Nogo-66-induced inhibition of neurite outgrowth and explore the potential mechanism. The expression of relative genes was detected by qRT-PCR and western blot. The function of FTX was determined by overexpression and knockdown techniques. The interaction between FTX and PDK1 was evaluated by RIP and RNA pull-down assays. FTX expression was downregulated by Nogo-66 in PC12 cells. Nogo-66-induced inhibition of neurite outgrowth was relieved by FTX overexpression. FTX bound to PDK1 protein to disturb the interaction between PDK1 and E3 ubiquitin ligase RNF126, thereby blocked the ubiquitination degradation of PDK1 and elevated PDK1 protein level. Mechanically, FTX involved in the Nogo-66-induced inhibition of neurite outgrowth through the PDK1/PKB/GSK-3β pathway. In SCI rats, FTX knockdown inhibited neurite outgrowth induced by the receptor antagonist of Nogo-66. The present results suggested that FTX took part in Nogo-66-inhibited neurite outgrowth, and FTX exerted its function through regulating PDK1/PKB/GSK-3β pathway.

Keywords: LncRNA FTX; Neurite outgrowth; Nogo-66; PDK1; PKB/GSK-3β.

Fig. 1

The effect of Nogo-66 on FXT. a PC12 cells were stimulated by NGF for 2 h and then treated with Nogo-66 (0, 0.1, 0.5, 1, and 2 μM) for 3 days. Relative expression of FTX was detected by qRT-PCR. **p < 0.01 vs. 0 μM Nogo-66 group. b PC12 cells were stimulated by NGF for 2 h and then treated with Nogo-66 (1 μM) for 0, 1, 3, 5, and 7 days. **p < 0.01 vs. 0 day group. The expression of FTX was determined by qRT-PCR. c PC12 cells were treated with 1 μM Nogo-66 and 1 μM NEP1-40 (a receptor antagonist of Nogo-66). 3 days after treatment, Relative expression of FTX was detected by qRT-PCR. **p < 0.01 vs. Control group; ^##p < 0.01 vs. Nogo-66+ PBS group. d SCI rats were treated with NEP1-40 (500 μM). Then the relative expression of FTX in spinal cord tissues was detected by qRT-PCR after 28 days. N = 7. **p < 0.01 vs. Sham group; ^##p < 0.01 vs. SCI + PBS group. PBS the solvent control of NEP1-40

Fig. 2

Neurite outgrowth under FXT overexpression. PC12 cells were stimulated by NGF for 2 h, treated with 1 μM Nogo-66 and then transfected with pcDNA-FTX or pcDNA. Cells were cultured for 3 days. a Images of neurite outgrowth (left) and the quantification of neurite length (right). Scale bar = 25 μm. **p < 0.01 vs. Control group; ^#p < 0.05 vs. Nogo-66+ pcDNA group. b The protein levels of PDK1, PKB, p-PKB, GSK-3β, and p-GSK-3β were detected by western blot. pcDNA the negative control of pcDNA-FTX

Fig. 3

FTX targeted PDK1. PC12 cells were used to perform the following experiments. a RNA pull-down: western blot was used to detect PDK1 in the FTX pulled-down complex. NC was the negative control of FTX. b RIP: PDK1 antibody was used for RIP. FTX was examined by qRT-PCR. IgG was the negative control of PDK1. Input: 4% cell lysate. **p < 0.01 vs. IgG. c–e PC12 cells were transfected with pcDNA-FTX or pcDNA. The protein level (c) and mRNA level (d) of FTX in PC12 cells were detected by qRT-PCR and western blot. e PC12 cells were treated with CHX (a protein synthesis inhibitor, 20 μg/mL) for 0, 50, 100 and 150 min. The protein level (left) and of PDK1 was detected by western blot. *p < 0.05, **p < 0.01 vs. pcDNA. pcDNA the negative control of pcDNA-FTX

Fig. 4

Effect of FTX overexpression on the ubiquitination of PDK1. a PC12 cells were transfected with pcDNA-FTX or pcDNA and then treated with proteasome inhibitor MG132 (10 μM) for 8 h. The protein level of PDK1 was determined by western blot. b 293 T cells were co-transfected with pcDNA-FTX (or pcDNA), Flag-PDK1 and HA. Cell lysates were utilized to IP using Flag antibody, followed by western blot using HA antibody (top). The whole-cell lysates (WCL) were subjected to detect PDK1 expression (bottom). c The interaction between PDK1 and RNF12 (E3 ubiquitin ligase) was shown by the Co-IP assay. Left: PC12 cells were co-transfected with PDK1, FLAG-RNF126, and pcDNA-FTX (or pcDNA). WCL was used to detect the expression of PDK1 (middle) and RNF126 (bottom) by western blot. Cell lysates were subjected to IP using FLAG antibody, followed by western blot using PDK1 antibody (top). Right: PC12 cells were transfected with pcDNA-FTX or pcDNA. Cell lysates were utilized to IP using PDK1 antibody, followed by western blot using RNF126 antibody. pcDNA the negative control of pcDNA-FTX. *p < 0.05, **p < 0.01

Fig. 5

FTX/PDK1 involved in Nogo-66 inhibiting neurite outgrowth. PC12 cells were stimulated with 100 ng/ml NGF for 2 h, treated with 1 μM Nogo-66, and transfected with pcDNA-FTX, shRNA-PDK1 or corresponding negative controls (pcDNA and shRNA). a Images of neurite outgrowth (left) and the quantification of neurite length (right). Scale bar = 25 μm. **p < 0.01 vs. control; ^#p < 0.05 vs. Nogo-66 + pcDNA group; ^$p < 0.05 vs. Nogo-66+ pcDNA-FTX+ shRNA group. b The protein levels of PDK1, p-PKB, PKB, GSK-3β, and p-GSK-3β were detected by western blot. pcDNA the negative control of pcDNA-FTX. shRNA the negative control of shRNA-PDK1

Fig. 6

Knockdown of FTX inhibited NEP1-40-induced neurite growth in SCI rats. SCI rats were treated with 500 μM NEP1-40 and intrathecally injected with LV-shRNA-FTX or LV-shRNA. a BBB scores of rats. b The expression of FTX was detected by qRT-PCR. c The protein levels of the neurite regeneration markers (GAP-43 and MAP-2) and neurite injury marker (APP) were detected by western blot. d The protein levels of PDK1, PKB, p-PKB, GSK-3β, p-GSK-3β were detected by western blot. n = 7. **p < 0.01 vs. PBS group; ^##p < 0.01 vs. NEP1-40+ shRNA group. PBS the solvent control of NEP1-40. pcDNA the negative control of pcDNA-FTX

Fig. 7

The diagram of the mechanism of this study