| Spinal cord injury(SCI)is a severe central nervous system damage.The patients of SCI need long-lasting and complex care.Etiologically,SCI can be divided into primary injury and secondary injury.Primary injury mainly refers to the mechanical injury caused by vertebral fracture or dislocation.The secondary injury occurs a few minutes after injury and lasts for weeks or months,causing lasting damage to the spinal cord.The inflammation response is extremely important in secondary SCI and accumulating attention has been paid to eliminating the inflammation after secondary SCI.Microglia is a traditional inflammatory cell in the central nervous system.Due to the destruction of blood vessels and an imbalance of homeostasis,microglia begin to activate and transform to pro-inflammatory M1 microglia or anti-inflammatory M2 microglia.Thus,decreasing the formation of M1 microglia in SCI will downregulate inflammation and reduce adverse effects on neurons.Circular RNAs(CircRNAs)form a covalent closed-loop structure with high levels of tissue-specific expression.Compared with linear RNAs,circRNAs are more stable and lack a poly-A tail or 5 ’cap.Our previous studies showed that circPrkcsh was markedly elevated in SCI tissues and was able to aggravate the inflammation of the astrocyte.In this study,we aimed to detect the expression,function,and mechanism of circPrkcsh in microglia.We used BV2 microglia,RAW.264.7 macrophage,HEK293T cell,and primary neuron in vitro studies.The inflammatory environment was induced by lipopolysaccharide(LPS).We transfected cells with overexpression plasmid,si-RNA,and lentivirus to upregulate or downregulate the target genes.The results of western blot assay,RT-qPCR,and immunofluorescence assay showed that upregulation of circPrkcsh could elevate the expression of protein and mRNA of M1 microglia,while upregulation of miR-488 showed an opposite trend.We proved that miR-488 could bind to circPrkcsh and MEKK1 was a target of miR-488 by bioinformatic prediction,RNA sequencing,Dualluciferase assay,Pulldown assay,and RIP assay.We constructed a standard mice SCI model through an SCI impactor and proved the success of surgery by HE staining.The FISH assay showed that circPrkcsh was upregulated in microglia of SCI mice compared with the sham group.At last,we injected mice with AAV-sh-circPrkcsh to downregulate the expression of circPrkcsh in SCI mice.We found that the inflammation responses of the spinal cord in mice were reduced in AAV-sh-circPrkcsh injected group compared with the control group.In summary,our research showed that circPrkcsh was increased after SCI and promoted the formation of M1 microglia.The downstream target of circPrkcsh was the miR-488/MEKK1 axis.Downregulation of circPrkcsh in SCI mice could alleviate inflammation and promote the functional recovery of the moto.Thus,our study shed light on further therapy of SCI. |
