PMC

shRNA silencing of PKIA-AS1 inhibits neuroinflammation by regulating CDK6 in SNL rats. (A) mRNA levels of IL-1β, IL-6, IL-12, and TNF-α in L5 spinal cord tissues in the rats 6 days after lentivirus infection. (B) The protein levels of IL-1β, IL-6, IL-12, and TNF-α in the L5 spinal cord of rats were tested by ELISA 6 days after lentivirus infection. N = 8 for each group; ^∗P < 0.05.

Overexpression of PKIA-AS1 is sufficient to cause pain behavior in rats. (A) Real-time PCR show that the expression of PKIA-AS1 in the control rats 6 days after infection of PKIA-AS1 lentivirus was significantly higher than that of the control group. ^∗P < 0.05. (B,C) Overexpression of PKIA-AS1 produced mechanical hyperalgesia (B) and thermal hyperalgesia (C) in the control rats. N = 8 for each group, ^∗P < 0.05. PWT, paw withdrawal threshold; PWL, paw withdrawal latency.

PKIA-AS1 modulated neuropathic pain by regulating CDK6. (A) CDK6 mRNA expression in SNL rat models infected with Lv-CDK6 6 days after lentivirus infection. (B) CDK6 protein expression in the spinal cord tissues of rats infected with indicated viruses. (C) The effect of CDK6 on mechanical allodynia was evaluated by PWT. (D) The effect of CDK6 on thermal hyperalgesia was assessed by PWL. N = 8 for each group; ^∗P < 0.05. PWT, paw withdrawal threshold; PWL, paw withdrawal latency.

Differentially expressed lncRNAs in spinal cord of SNL rats. (A,B) Spinal never ligation induced mechanical hyperalgesia (A) and thermal hyperalgesia (B) in rats. N = 8 for each group, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. control group. (C) Volcano plot shown the downregulated and upregulated lncRNAs. (D) Real-time PCR data confirmed the expressions of top 10 upregulated lncRNAs in spinal cord of SNL rats and control group. ^∗P < 0.05 vs. control group. SNL, spinal never ligation; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; lncRNA, long non-coding RNA.

lncRNA PKIA-AS1 expression is increased in the SNL spinal cord and contributes to the SNL-induced neuropathic pain. (A) Real-time PCR data show the expressions of PKIA-AS1 in the SNL spinal cord on different time points. (B) Real-time PCR data show that the expressions of PKIA-AS1 in the SNL spinal cord treated with shPKIA-AS1 lentivirus were significantly lower than that of the SNL+Lv-NC group. (C,D) shRNA silencing of PKIA-AS1 alleviates mechanical hyperalgesia (C) and thermal hyperalgesia (D) in the SNL rats. N = 8 for each group, ^∗P < 0.05 vs. control group. SNL, spinal never ligation; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; lncRNA, long non-coding RNA.

shRNA silencing of PKIA-AS1 repressed neuroinflammation in SNL rats. (A) Immunofluorescence assay results show that silencing of PKIA-AS1 repressed SNL-mediated astrocyte and microglia activation evaluated by GFAP and Iba-1, respectively. Scale bar: 100 μm, N = 8 for each group. (B) mRNA levels of IL-1β, IL-6, IL-12, and TNF-α in L5 spinal cord tissues in the rats 6 days after infection of PKIA-AS1 lentivirus. (C) The protein levels of IL-1β, IL-6, IL-12, and TNF-α in the L5 spinal cord of rats were tested by ELISA at postoperative day 6. N = 8 for each group; ^∗P < 0.05.

PKIA-AS1 epigenetically regulates CDK6 by hypomethylating its promotor. (A) The differential protein spots from 2-DE gels by differential proteomics analysis. The gel was stained with coomassie brilliant blue. 2-DE, two-dimensional polyacrylamide gel electrophoresis. (B) CDK6 mRNA expression in PC12 cells infected with Lv-PKIA-AS1. (C) CDK6 protein expression in PC12 cells infected with Lv-PKIA-AS1. (D) Dual luciferase assay shown that PKIA-AS1 induced CDK6 promotor activity. (E) The correlation between PKIA-AS1, CDK6 promotor and Ago2 was detected by RIP assay. Cellular lysates were immunoprecipitated using Ago2 antibody or IgG. PKIA-AS1 and CDK6 promotor expression was tested by qRT-PCR. (F) PC12 cells were infected with PKIA-AS1 lentivirus. qMSP was perform to analyze CDK6 promoter methylation. (G) DNMT1, DNMT3a, and DNMT3b protein levels were assessed by western blot in PC12 cells infected with Lv-PKIA-AS1; ^∗P < 0.05.