The Mechanism Of CIRCpedia＿4214 Regulates Microglia Pyroptosis After Spinal Cord Injury Via MiR-212-5p/Myd88 Axis

Background:Spinal cord injury（SCI）is one of the most disabling and destructive diseases of the central nervous system,which seriously affects the quality of life and physical and mental health of patients and impose a huge economic burden on families and society,therefore,the treatment of SCI is of great social significance.Pyroptosis is a new type of programmed cell death that accompanies the inflammatory response and has both necrotic and apoptotic morphological characteristics.Microglia are the most important immune inflammatory cells in the central nervous system.In the mechanism of SCI,microglia pyroptosis-mediated neuroinflammatory response directly affects the recovery of neurological function.Circ RNA is a circular non-coding RNA molecule commonly found in eukaryotes and is capable of regulating gene transcription or post-transcriptional level.To date,no circ RNA has been reported in the literature to regulate microglial pyroptosis in SCI.Our previous circ RNA microarray showed that CIRCpedia₄214 was relatively highly expressed in the rat model of SCI,and knockdown of CIRCpedia₄214 promoted macrophage M2-type polarization and reduced inflammation.This study aims to elucidate the role of CIRCpedia₄214 on microglia pyroptosis in SCI and the related molecular mechanism,and provide a new theoretical basis for the treatment of SCI.Materials and Methods:1.qRT-PCR was used to detect the expression level of CIRCpedia₄214 in SCI spinal cord tissue and microglia（GMI-R1）pyroptosis model.Sanger sequencing,cytosolic RNA isolation and extraction assay,and RNase R digestion assay were used to verify the cyclic properties and sublocalization of CIRCpedia₄214 in the cells.The transfection efficiency was examined by transfecting CIRCpedia₄214 overexpression plasmid and small interfering RNA into GMI-R1 cell pyroptosis model by qRT-PCR.Finally,changes in expression levels of pyroptosis-related genes and proteins were detected by qRT-PCR,Western Blot and immunofluorescence,and changes in concentrations of related inflammatory factors were detected by ELISA.2.Prediction of miRNAs and m RNAs that may be associated with CIRCpedia₄214 using multiple online bioinformatics prediction website.CIRCpedia₄214 WT,CIRCpedia₄214 Mut,Myd88 3’UTR WT,Myd88 3’UTR Mut,miR-212-5p mimics,miR-212-5p inhibitors,Myd88 overexpression plasmids and each negative control group were constructed and transfected two-by-two into GMI-R1 cells,and the target binding between miR-212-5p and CIRCpedia₄214,miR-212-5p and Myd88 was verified by dual luciferase reporter assay.Finally,changes in expression levels of pyroptosis-related genes and proteins were detected by qRT-PCR,Western Blot,and immunofluorescence,and changes in concentrations of related inflammatory factors were detected by ELISA.3.In vivo experiments were performed using chemically modified si RNA interference technique with intrathecal injections every 3 days preoperatively for 2 weeks before establishing the SCI model.CIRCpedia₄214,miR-212-5p,Myd88 m RNA,pyroptosis-related genes and protein expression were detected by qRT-PCR,Western Blot or immunofluorescence in Sham group,SCI group,SCI+si NC group and SCI+si RNA group.Motor function recovery in rats was assessed using motor function score（Basso,Beattie & Bresnahan locomotor rating scale）and Louisville swimming scale.Nissl staining was used to detect neuronal loss.Results:1.CIRCpedia₄214 expression was significantly elevated in spinal cord tissue（n=21）in the SCI group compared to the sham group（n=21）;similarly,CIRCpedia₄214 expression was significantly elevated in the GMI-R1 cell pyroptosis model compared to the control group（P < 0.05）.The expression of CIRCpedia₄214 was significantly up-regulated（P < 0.05）in GMI-R1 cell pyroptosis model by transfection of CIRCpedia₄214 overexpression plasmid compared with the control group.m RNA and protein expression levels associated with GMI-R1 cell pyroptosis were down-regulated（P < 0.05）in the CIRCpedia₄214 overexpression group compared with the control group;The cell supernatant inflammatory factors（IL-1β,IL-18）concentrations were significantly higher（P < 0.05）compared to the control group.In contrast,silencing CIRCpedia₄214 produced the opposite effect（P < 0.05）.2.Bioinformatics online website predicts the possible target miRNAs bound by CIRCpedia₄214,including miR-336-3p,miR-10b-5p,miR-212-5p,miR-135b-3p,miR-141-5p and miR-667-5p.After CIRCpedia₄214 expression was interfered,GMI-R1 cells showed the most significant upregulation of miR-212-5p expression（P < 0.05）.Expression of miR-212-5p was significantly downregulated（P < 0.05）in spinal cord tissue of SCI group（n=21）compared to sham group（n=21）.Dual luciferase reporter assay confirmed that CIRCpedia₄214 interacts with miR-212-5p.In vitro experiments confirmed that miR-212-5p mimics could partially reverse the GMI-R1 cell scorching and inflammatory factor（IL-1β,IL-18）secretion promotion caused by overexpression of CIRCpedia₄214（P < 0.05）.3.Combined with bioinformatics online website and literature reports,miR-212-5p was predicted to possibly directly bind to Myd88.Mi R-212-5p expression was interfered,and Myd88 expression was upregulated in GMI-R1 cells（P < 0.05）.Myd88 expression was significantly elevated（P < 0.05）in spinal cord tissue（n=21）in the SCI group compared to the sham group（n=21）.Dual luciferase reporter assay confirmed that Myd88 could be directly targeted with miR-212-5p.In vitro experiments confirmed that Myd88 overexpression plasmid could partially reverse the inhibition of GMI-R1 cell scorching and inflammatory factor（IL-1β,IL-18）secretion caused by miR-212-5p mimics（P < 0.05）.In addition,miR-212-5p inhibitors partially reversed the down-regulation of Myd88 expression caused by knockdown of CIRCpedia₄214（P < 0.05）.4.In vivo experimental studies showed that m RNA expression of CIRCpedia₄214 and Myd88 were significantly down-regulated and miR-212-5p expression was up-regulated in the si RNA group compared to the si NC group（P < 0.05）;furthermore,both microglia pyroptosis and neuronal loss were reduced after knockdown of CIRCpedia₄214,and motor function was significantly improved in rats（P <0.05）.Conclusion:We demonstrated that CIRCpedia₄214 expression was upregulated in rat microglia after SCI and promotes microglia pyroptosis,and which was regulated by the CIRCpedia₄214/miR-212-5p/Myd88 axis.In vivo interference with CIRCpedi a₄214 inhibited microglia pyroptosis and improved motor function after SCI in rats,providing a new theoretical basis and potential therapeutic target for the treatment of SCI.