Down-regulated miR-448 relieves spinal cord ischemia/reperfusion injury by up-regulating SIRT1

Abstract

MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII). The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.

Figure 1.. Altered expression of miR-448 and SIRT1 in ischemia/reperfusion injury (SCII) tissues. A, Expression of miR-448 in control and SCII tissues was quantified by qRT-PCR (n=12). B, Expressions of SIRT1 mRNA and protein were analyzed by qRT-PCR and western blot (n=12), respectively. C, Hind limb motor function was assessed by Basso, Beattie, and Bresnahan (BBB) scoring. Data are reported as means±SD. *P<0.05 vs control (t-test).

Figure 2.. Hypoxia treatment altered the expression of miR-448 and SIRT1 in nerve cells. A, Expression of miR-448 in AGE1.HN and PC12 cells with or without hypoxia treatment was determined by qRT-PCR. B, Expressions of SIRT1 mRNA and protein in AGE1.HN and PC12 cells with or without hypoxia treatment were analyzed by qRT-PCR and western blot, respectively. Data are reported as means±SD. *P<0.05 vs control (t-test).

Figure 3.. Hypoxia treatment regulated the expression of SIRT1 through miR-448. A, Expression of miR-448 in AGE1.HN and PC12 cells in control or treated with hypoxia, or transfected with NC or miR-448 inhibitor was determined by qRT-PCR. B, Expressions of SIRT1 mRNA and protein in AGE1.HN and PC12 cells in control or treated with hypoxia or transfected with NC or miR-448 inhibitor were analyzed by qRT-PCR and western blot, respectively. Data are reported as means±SD. *P<0.05 vs control. ^#P<0.05 vs NC (t-test). NC: negative control.

Figure 4.. MiR-448 participated in the regulation of nerve cell apoptosis. A, Cell apoptosis was analyzed by flow cytometry in AGE1.HN and PC12 cells treated with hypoxia, or transfected with NC or miR-448 inhibitor. *P<0.05 vs control. ^#P<0.05 vs NC (t-test). B, Cell apoptosis was analyzed by flow cytometry in AGE1.HN and PC12 cells transfected with pre-NC or miR-448 mimic. Data are reported as means±SD. *P<0.05 vs pre-NC (t-test). NC: negative control.

Figure 5.. Expression of SIRT1 was regulated by miR-448 in AGE1.HN cells. A, Predicted binding region of miR-448 and the 3′-UTR of WT-SIRT1, and sequence of the mut-SIRT1 3′-UTR. B, Relative luciferase activity of WT-SIRT1 and mut-SIRT1 in AGE1.HN cells transfected with NC or miR-448 inhibitor. Expressions of SIRT1 mRNA and protein in AGE1.HN cells transfected with NC or miR-448 inhibitor were analyzed by qRT-PCR and western blot, respectively. *P<0.05 vs NC (t-test). C, Relative luciferase activity of WT-SIRT1 and mut-SIRT1 in AGE1.HN cells transfected with pre-NC or miR-448 mimic. Expressions of SIRT1 mRNA and protein in AGE1.HN cells transfected with pre-NC or miR-448 mimic were analyzed by qRT-PCR and western blot, respectively. Data are reported as means±SD. ^#P<0.05 vs pre-NC (t-test). NC: negative control.

Figure 6.. Hypoxia treatments regulated the expression of SIRT1 through miR-448 for participating in nerve cell apoptosis. A, Cell apoptosis analyzed by flow cytometry in AGE1.HN and PC12 cells treated with hypoxia, and transfected or co-transfected with NC or miR-448 inhibitor or si-control or si-SIRT1. *P<0.05 vs NC. ^#P<0.05 vs si-control (t-test). B, Cell apoptosis analyzed by flow cytometry in AGE1.HN and PC12 cells transfected with pre-NC or miR-448 mimic. Data are reported as means±SD. *P<0.05 vs pre-NC. ^#P<0.05 vs pcDNA (t-test). NC: negative control.

Figure 7.. Impact of miR-448 expression level on neurological function of rats with ischemia/reperfusion injury (SCII). A, Motor deficit index (MDI) score and the Basso, Beattie, and Bresnahan (BBB) score. B, Expressions of miR-448, SIRT1 mRNA and protein in spinal tissues transfected with NC (n=10) or miR-448 inhibitor (n=10) analyzed by qRT-PCR and western blot, respectively. Data are reported as means±SD. *P<0.05 vs NC (t-test). NC: negative control.

Figure 8.. Flowchart illustrating the different findings reported on the neuroprotective effect of SRIT1 by suppressing neuronal apoptosis in ischemia/reperfusion injury (SCII). SCI: spinal cord injury.