LncRNA KCNQ1OT1 promotes the apoptosis and inflammatory response of microglia by regulating the miR-589-5p/NPTN axis after spinal cord injury

Spinal cord injury (SCI) is a devastating traumatic condition accompanied with excessive inflammatory response and apoptosis of microglia. Long noncoding RNAs (lncRNAs) have been confirmed to be key regulators of cell inflammatory response. Nevertheless, the role of lncRNA KCNQ1OT1 in microglia apoptosis or inflammatory response after SCI remains to be explored. Our study focused on exploring the role and mechanism of KCNQ1OT1 in microglia after SCI. RT-qPCR showed that SCI induced the increase of KCNQ1OT1 level in mice spinal cord. Inhibition of KCNQ1OT1 suppressed the inflammatory response and apoptosis of microglia. In addition, KCNQ1OT1 was proved to bind with miR-589-5p, and NPTN was directly targeted by miR-589-5p. Furthermore, KCNQ1OT1 was negatively correlated with miR-589-5p and positively associated with NPTN. Rescue assays indicated that NPTN overexpression reversed the anti-inflammatory and anti-apoptosis effects of KCNQ1OT1 silencing. In summary, these data revealed that KCNQ1OT1 promoted inflammatory response and apoptosis of microglia by regulating the miR-589-5p/NPTN axis after SCI, which may offer a novel promising therapeutic target for SCI.

Key words
KCNQ1OT1; miR-589-5p; NPTN; spinal cord injury

Authorship

ZHAOMING CHU

Department of Orthopedics, The First People’s Hospital of Lianyungang, No 6. Zhenhua East Road, Lianyungang 222000, Jiangsu, ChinaDepartment of OrthopedicsChinaJiangsu, ChinaDepartment of Orthopedics, The First People’s Hospital of Lianyungang, No 6. Zhenhua East Road, Lianyungang 222000, Jiangsu, China

https://orcid.org/0000-0001-8118-0493

YOU LU

https://orcid.org/0000-0003-0244-5161

RUJIE QIN

https://orcid.org/0000-0001-9671-7927

YUEFU DONG

https://orcid.org/0000-0002-4895-9339

Correspondence to: Yuefu Dong E-mail: YuefuDong0229@outlook.com

AUTHOR CONTRIBUTIONS

Zhaoming Chu and Yuefu Dong conceived of the presented idea. Zhaoming Chu, You Lu, Rujie Qin, and Yuefu Dong developed the theory and performed the computations. Zhaoming Chu and Yuefu Dong verified the analytical methods. Zhaoming Chu and Yuefu Dong supervised the findings of this work. All authors discussed the results and contributed in the final manuscript. The authors report no declarations of interest.

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Figures

Figures (5)

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Figure 1
SCI induced the increase of the cell apoptosis and inflammation. (a) The behavioral analyses of the mice were measured via Basso, Beattie, and Bresnahan (BBB) scores. (b) Cell apoptosis in the SCI group and sham group was examined by use of TUNEL assay. (c) Western blot analysis was employed to evaluate the protein levels of apoptosis associated genes (Bax, Bcl-2 and Cleaved-caspase-3). (d-f) ELISA was performed to detect the concentrations of proinflammatory cytokines (IL-6, TNF-α and IL-1β) in the mouse serum. N=5 in each group. *p < 0.05. **p < 0.01.

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Figure 2
Silencing of KCNQ1OT1 alleviated apoptosis of microglia and inflammatory response. (a) RT-qPCR analysis was carried out to detect KCNQ1OT1 level after injection of LV-shRNA targeting KCNQ1OT1 into mice. (b) BBB scores showed the behavioral analyses of the mice. (c-d) TUNEL staining was used to test the number of TUNEL positive cells in different groups. (e-f) The protein levels of Bax, Bcl-2 and Cleaved-caspase-3 in different groups were determined by western blot analysis. (g-i) ELISA was used to measure the concentrations of IL-6, TNF-α and IL-1β in different groups. N=8 in each group. *p < 0.05. **p < 0.01.

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Figure 3
KCNQ1OT1 bound with miR-589-5p. (a) The expression of miR-589-5p, miR-146a-5p and miR-98-5p in SCI mice was detected by RT-qPCR. (b) RIP revealed KCNQ1OT1 and miR-589-5p were in the same RNA-induced silencing complex (RISC). (c) RT-qPCR displayed the expression of miR-589-5p in microglia from the SCI mice after transfection of miR-589-5p mimics. (d) The putative binding site between KCNQ1OT1 and miR-589-5p was exhibited. (e) Luciferase reporter assay showed KCNQ1OT1 bound with miR-589-5p. (f) RT-qPCR delineated miR-589-5p expression after LV-miR-589-5p injection into SCI mice. (g) Pearson’s correlation analysis unveiled the correlation between expression of miR-589-5p and KCNQ1OT1 expression in SCI mice. (h) BBB scores was used to analyze the neurological function of the mice. (i-j) The number of TUNEL positive cells in different groups was measured by TUNEL staining. (k-m) ELISA was used to measure the concentrations of IL-6, TNF-α and IL-1β in different groups. N=8 in each group for h-m. *p < 0.05. **p < 0.01.

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Figure 4
NPTN was a target of miR-589-5p. (a) Bioinformatics analysis was performed to find the potential targets of miR-589-5p. (b) The expression of the candidate mRNAs upon miR-589-5p mimics was detected by RT-qPCR. (c) RIP indicated the enrichment of miR-589-5p and NPTN in Ago2 and IgG group. (d) The potential binding site between NPTN and miR-589-5p was exhibited. Luciferase reporter assay showed miR-589-5p bound with NPTN. (e) NPTN level in spinal cord tissues. (f-g) Pearson’s correlation analysis showed the correlation between NPTN and miR-589-5p as well as NPTN and KCNQ1OT1. *p < 0.05. **p < 0.01.

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Figure 5
KCNQ1OT1 promoted the apoptosis of microglia and inflammatory response by regulating NPTN in SCI. (a) RT-qPCR and western blot analyses disclosed the mRNA and protein levels of NPTN in SCI mice after injection of LV-NPTN. (b-c) The number of TUNEL positive cells in different groups was assessed by TUNEL staining. (d) The protein levels of Bax, Bcl-2 and Cleaved-caspase-3 in SCI mice were detected by western blot analysis. (e) ELISA measured the concentrations of IL-6, TNF-α and IL-1β in different groups. N=8 in each group. *p < 0.05. **p < 0.01.

Figure 1 SCI induced the increase of the cell apoptosis and inflammation. (a) The behavioral analyses of the mice were measured via Basso, Beattie, and Bresnahan (BBB) scores. (b) Cell apoptosis in the SCI group and sham group was examined by use of TUNEL assay. (c) Western blot analysis was employed to evaluate the protein levels of apoptosis associated genes (Bax, Bcl-2 and Cleaved-caspase-3). (d-f) ELISA was performed to detect the concentrations of proinflammatory cytokines (IL-6, TNF-α and IL-1β) in the mouse serum. N=5 in each group. *p < 0.05. **p < 0.01.

Figure 2 Silencing of KCNQ1OT1 alleviated apoptosis of microglia and inflammatory response. (a) RT-qPCR analysis was carried out to detect KCNQ1OT1 level after injection of LV-shRNA targeting KCNQ1OT1 into mice. (b) BBB scores showed the behavioral analyses of the mice. (c-d) TUNEL staining was used to test the number of TUNEL positive cells in different groups. (e-f) The protein levels of Bax, Bcl-2 and Cleaved-caspase-3 in different groups were determined by western blot analysis. (g-i) ELISA was used to measure the concentrations of IL-6, TNF-α and IL-1β in different groups. N=8 in each group. *p < 0.05. **p < 0.01.

Figure 3 KCNQ1OT1 bound with miR-589-5p. (a) The expression of miR-589-5p, miR-146a-5p and miR-98-5p in SCI mice was detected by RT-qPCR. (b) RIP revealed KCNQ1OT1 and miR-589-5p were in the same RNA-induced silencing complex (RISC). (c) RT-qPCR displayed the expression of miR-589-5p in microglia from the SCI mice after transfection of miR-589-5p mimics. (d) The putative binding site between KCNQ1OT1 and miR-589-5p was exhibited. (e) Luciferase reporter assay showed KCNQ1OT1 bound with miR-589-5p. (f) RT-qPCR delineated miR-589-5p expression after LV-miR-589-5p injection into SCI mice. (g) Pearson’s correlation analysis unveiled the correlation between expression of miR-589-5p and KCNQ1OT1 expression in SCI mice. (h) BBB scores was used to analyze the neurological function of the mice. (i-j) The number of TUNEL positive cells in different groups was measured by TUNEL staining. (k-m) ELISA was used to measure the concentrations of IL-6, TNF-α and IL-1β in different groups. N=8 in each group for h-m. *p < 0.05. **p < 0.01.

Figure 4 NPTN was a target of miR-589-5p. (a) Bioinformatics analysis was performed to find the potential targets of miR-589-5p. (b) The expression of the candidate mRNAs upon miR-589-5p mimics was detected by RT-qPCR. (c) RIP indicated the enrichment of miR-589-5p and NPTN in Ago2 and IgG group. (d) The potential binding site between NPTN and miR-589-5p was exhibited. Luciferase reporter assay showed miR-589-5p bound with NPTN. (e) NPTN level in spinal cord tissues. (f-g) Pearson’s correlation analysis showed the correlation between NPTN and miR-589-5p as well as NPTN and KCNQ1OT1. *p < 0.05. **p < 0.01.

Figure 5 KCNQ1OT1 promoted the apoptosis of microglia and inflammatory response by regulating NPTN in SCI. (a) RT-qPCR and western blot analyses disclosed the mRNA and protein levels of NPTN in SCI mice after injection of LV-NPTN. (b-c) The number of TUNEL positive cells in different groups was assessed by TUNEL staining. (d) The protein levels of Bax, Bcl-2 and Cleaved-caspase-3 in SCI mice were detected by western blot analysis. (e) ELISA measured the concentrations of IL-6, TNF-α and IL-1β in different groups. N=8 in each group. *p < 0.05. **p < 0.01.

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LncRNA KCNQ1OT1 promotes the apoptosis and inflammatory response of microglia by regulating the miR-589-5p/NPTN axis after spinal cord injury

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[1] Correspondence to: Yuefu Dong E-mail: YuefuDong0229@outlook.com