| Objective: Spinal cord ischaemia reperfusion injury(SCIRI)is one of the most severe complications of thoracic and abdominal aortic surgery,which results in loss of sensory and motor function below the injury level,causing significant physical,emotional,and economic burdens for patients and society.Although various preventive and therapeutic strategies have been applied,the outcomes remain unsatisfactory.An in-depth understanding of the molecular mechanisms underlying SCIRI and effective strategies to prevent irreversible neuronal death are needed to improve the therapeutic outcomes.Non-coding RNAs(nc RNAs)are functional molecules that do not encode proteins but have diverse regulatory functions in organisms.Long non-coding RNAs(lnc RNAs)can act as molecular sponges by competitively binding to micro RNAs(mi RNAs)and participating in the SCIRI process.In our previous experiments,we found differential expression of plasmacytoma variant translocation 1(PVT1)in a rat model of spinal cord ischemia-reperfusion injury.PVT1 has been reported to regulate inflammation and cell apoptosis in brain ischemia-reperfusion injury by targeting mi RNAs.Bioinformatics analysis revealed a potential binding relationship between PVT1 and mi R-210-5p,which negatively regulates target gene expression and participates in inflammation,angiogenesis,and cell apoptosis in the central nervous system.Furthermore,phosphatidylinositol-3-kinase regulatory subunit 3(PIK3R3)was predicted to be a target gene of mi R-210-5p,which regulates neuronal apoptosis by activating protein kinase B(AKT)in central nervous system diseases.However,their roles and interactions in spinal cord ischemia-reperfusion injury have not been reported.This study aims to explore the roles and regulatory mechanisms of PVT1,mi R-210-5p,and PIK3R3 in spinal cord ischemia-reperfusion injury,providing new targets and perspectives for the clinical treatment of SCIRI.Methods:1.SCIRI animal model was established by clamping the rat aortic arch for 14 minutes.The m RNA expression levels of lnc RNA PVT1 in rats were detected by quantitative real-time polymerase chain reaction(q RT-PCR)at 6h,12 h,24h,and 48 h after SCIRI.Bioinformatics analysis was used to identify the mi RNAs bound by PVT1.The m RNA expression levels of mi R-210-5p at different time points were measured by q RT-PCR,and the time point of 48 h post-reperfusion was selected for subsequent studies based on the experimental results.PVT1 overexpression lentivirus was injected into the rat intrathecal space for 7 days before modeling,and the Tarlov score was used to evaluate the rat’s motor function.The Evan’s blue(EB)staining method was used to evaluate blood-spinal cord barrier(BSCB)permeability,and q RT-PCR was used to detect changes in PVT1 and mi R-210-5p m RNA expression after PVT1 overexpression.The dual-luciferase reporter gene assay was used to detect the targeted binding relationship between PVT1 and mi R-210-5p.2.An animal model of SCIRI was established by clamping the rat’s aortic arch for14 minutes.q RT-PCR was used to measure the m RNA expression of PIK3R3 at different time points after SCIRI in rats.A dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between mi R-210-5p and PIK3R3.Seven days before modelling,PVT1 overexpression lentivirus was injected into the rat spinal cord via intrathecal injection.The protein expression levels of PIK3R3,p-AKT,Bcl-2,Beclin-1,Bax,and cleaved caspase-3 in the spinal cord tissues of rats from different groups after PVT1 overexpression were measured by Western blot(WB).TUNEL assay was used to detect the apoptosis response of spinal cord cells in different groups of rats.Rats were injected with mi R-210-5p and/or PVT1 overexpression lentivirus before modelling,and their neuro-motor function was evaluated by behavioral tests.The BSCB permeability of rats was assessed by Evans Blue staining.q RT-PCR was employed to measure the expression levels of mi R-210-5p in different groups of rat spinal cord tissues.WB was used to determine the protein expression levels of PIK3R3,p-AKT,Bcl-2,Beclin-1,Bax,and cleaved caspase-3 in the spinal cord tissues of rats from different groups.TUNEL assay was used to detect the apoptosis response of spinal cord cells in different groups of rats.3.The researchers established an oxygen-glucose deprivation/reperfusion(OGD/R)model in PC12 cells and observed changes in cell morphology at different time points after reperfusion using optical microscopy.Hochest 33342 staining was used to assess cell apoptosis at different time points.q RT-PCR was utilized to measure the m RNA expression levels of PVT1 at different time points after OGD/R.Based on the experimental results,the researchers selected 24 h after reperfusion as the time point for subsequent research.The m RNA expression levels of mi R-210-5p and PIK3R3 in cells at24 h after reperfusion were measured using q RT-PCR.Prior to constructing the OGD/R model,the researchers transfected cells with GV658 CMV-PVT1 plasmids for 48 hours to overexpress PVT1.The m RNA expression levels of PVT1,mi R-210-5p,and PIK3R3 in cells from different groups were measured using q RT-PCR,and the protein expression levels of PIK3R3,p-AKT,Bcl-2,Beclin-1,Bax,and cleaved caspase-3 were assessed using WB.Furthermore,the researchers transfected cells with mi R-210-5p mimic or mi R-210-5p inhibitor plasmids for 48 hours to overexpress or silence mi R-210-5p,respectively.The m RNA expression levels of mi R-210-5p and PIK3R3 in cells from different groups were measured using q RT-PCR,and the protein expression levels of PIK3R3,p-AKT,Bcl-2,Beclin-1,Bax,and cleaved caspase-3 were assessed using WB.Additionally,the researchers transfected cells with si PVT1 or si PVT1+mi R-210-5p inhibitor plasmids for 48 hours and measured the m RNA expression levels of PIK3R3 in cells from different groups using q RT-PCR.The protein expression levels of PIK3R3,p-AKT,Bcl-2,Beclin-1,Bax,and cleaved caspase-3 were also assessed using WB.Finally,cell apoptosis in different groups was evaluated using TUNEL staining.Result:1.The qRT-PCR analysis showed a significant decrease in the expression level of PVT1 m RNA in the spinal cord tissue of rats after 48 hours of spinal cord ischemia-reperfusion injury(SCIRI).Bioinformatics network prediction revealed a good binding relationship between PVT1 and mi R-210-5p.Furthermore,q RT-PCR analysis showed a significant increase in the expression level of mi R-210-5p in rat spinal cord tissue 48 hours after reperfusion.Seven days before modeling,intrathecal injection of PVT1 overexpressing lentivirus was performed,and Tarlov scores indicated that overexpression of PVT1 could significantly improve rat hind limb motor function.EB staining also showed that overexpression of PVT1 could improve BSCB permeability.q RT-PCR analysis demonstrated a significant increase in the expression level of PVT1 in rat spinal cord tissue after intrathecal injection of PVT1 overexpressing lentivirus,and at the same time,the expression level of mi R-210-5p was significantly downregulated.Dual-luciferase reporter gene assay verified the targeted binding relationship between PVT1 and mi R-210-5p.2.The qRT-PCR results showed that the m RNA expression level of PIK3R3 in rat spinal cord tissue significantly decreased after 48 h of reperfusion following SCIRI.The dual-luciferase reporter gene assay confirmed the targeting relationship between mi R-210-5p and PIK3R3.Seven days before modelling,PVT1 overexpression lentivirus was injected into the intrathecal space,which significantly increased the protein expression levels of PIK3R3,p-AKT,and Bcl-2,while downregulating the expression levels of Beclin-1,Bax,and cleaved caspase-3.TUNEL staining revealed that the number of apoptotic cells in the spinal cord tissue of the SCIRI group was significantly higher than that of the Sham group,and overexpression of PVT1 significantly reduced the number of apoptotic cells in the rat spinal cord.mi R-210-5p or/and PVT1 overexpression lentivirus were injected into the intrathecal space of rats seven days before modelling,and the Tarlov score showed that overexpression of mi R-210-5p impaired hind limb motor function in rats.EB staining revealed that overexpression of mi R-210-5p disrupted BSCB permeability in rats,and overexpression of mi R-210-5p reversed the improvement function of PVT1 overexpression.q RT-PCR measurements showed that the m RNA expression level of mi R-210-5p in the PVT1+mi R-210-5p group was significantly higher than that in the PVT1 group.After overexpression of mi R-210-5p,the protein expression levels of PIK3R3,p-AKT,and Bcl-2 significantly decreased,while the expression levels of Beclin-1,Bax,and cleaved caspase-3 significantly increased.Overexpression of mi R-210-5p reversed the protein regulatory effect of PVT1 overexpression.TUNEL staining showed that overexpression of mi R-210-5p exacerbated spinal cord cell apoptosis,while overexpression of mi R-210-5p reversed the inhibitory effect of PVT1 on spinal cord cell apoptosis.3.After OGD/R in PC12 cells,cell morphology significantly changed,the number of cells decreased significantly,and the nuclear volume increased and became round.Hochest 33342 stainings showed that apoptosis gradually increased,and apoptosis was significantly higher in the 24-hour reoxygenation group than in the control group.Apoptotic bodies appeared after 48 hours of reoxygenation.The expression level of PVT1 significantly decreased after 24 hours of reoxygenation,while the expression of mi R-210-5p significantly increased,and the expression level of PIK3R3 significantly decreased.Overexpression of PVT1 significantly reduced the expression level of mi R-210-5p in cells,while the expression level of PIK3R3 significantly increased.After overexpression of PVT1,the protein expression levels of PIK3R3,p-AKT,and Bcl-2significantly increased,while the protein expression levels of Bax,cleaved caspase-3,and Beclin-1 significantly decreased.TUNEL staining showed that the number of apoptotic cells significantly decreased after overexpression of PVT1.After overexpression of mi R-210-5p,the m RNA expression level of PIK3R3 significantly decreased,and the protein expression levels of PIK3R3,p-AKT,and Bcl-2 significantly decreased,while the protein expression levels of Bax,cleaved caspase-3,and Beclin-1significantly increased.TUNEL staining showed that the number of apoptotic cells significantly increased after overexpression of mi R-210-5p,and overexpression of mi R-210-5p reversed the anti-apoptotic effect of PVT1 overexpression.Silencing mi R-210-5p achieved the opposite regulatory results.After silencing PVT1,the m RNA expression level of PIK3R3 significantly decreased,and the protein expression levels of PIK3R3,p-AKT,and Bcl-2 significantly decreased,while the protein expression levels of Bax,cleaved caspase-3,and Beclin-1 significantly increased.Silencing mi R-210-5p reversed the regulatory effect of si PVT1.Conclusion: In the rat model of SCIRI and the PC12 cell OGD/R model,PVT1 was found to target mi R-210-5p,and overexpression of PVT1 downregulated the level of mi R-210-5p.Mi R-210-5p was found to negatively regulate the expression of PIK3R3 through direct targeting,and PVT1 competitively bound to mi R-210-5p to upregulate the expression of PIK3R3,activate the AKT signaling pathway,and alleviate cell apoptosis response after SCIRI and cell OGD/R,thereby improving rat hind limb motor function and blood-spinal cord barrier permeability,and exhibiting spinal cord protective function. |
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