The Mechanism Of LncRNA NONRATT009773.2 In The Spinal Cord Regulating Bone Cancer Pain Through MiR-708-5p/CXCL13 Axis

Objective:Bone cancer pain(BCP)is a chronic pain state caused by malignant tumor metastasis to bone.It is one of the most common clinical symptoms in patients with advanced tumor.The mechanism of BCP has not been clarified,and there is a lack of effective treatment in clinic.Recent studies have shown that long noncoding RNA(lncRNA)plays a variety of important biological functions.The purpose of this study is to investigate the role and molecular mechanism of lncRNA NONRATT009773.2 in the spinal cord of BCP rats,and to provide a new theoretical basis for the prevention and treatment of BCP.Methods:1.We looked for the lncRNA high-throughput sequencing data of BCP rats’spinal cord tissue in GEO database and screened the significantly differentially expressed lncRNA in the spinal cord of BCP rats through bioinformatics analysis.2.BCP model was established,and behavioral tests were used to determine the degree of mechanical allodynia and heat hyperalgesia.3.In order to verify the results of bioinformatics analysis,the expression of lncRNA NONRATT009773.2 in the spinal cord of BCP rats was detected by RT-PCR.4.Lentivirus vectors were used to silence or overexpress lncRNA NONRATT009773.2 in the spinal cord of rats,and the pain behavior of rats was detected.5.The cellular localization of lncRNA NONRATT009773.2 in the spinal cord was detected by fluorescence in situ hybridization and immunofluorescence staining.6.The miRNA binding to lncRNA NONRATT009773.2 was screened by bioinformatics,and miR-708-5p was selected.The double luciferase reporter gene experiment verified the relationship between them.7.The mRNA binding to miR-708-5p was screened by bioinformatics,and CXCL13 was selected.The double luciferase reporter gene experiment verified the relationship between them.8.The interaction between lncRNA NONRATT009773.2,miR-708-5p and CXCL13 was verified by RT-PCR at the animal level.9.RT-PCR and ELISA were used to detect the expression of CXCL13 mRNA and protein in the spinal cord of BCP rats.10.Silenced CXCL13 in the spinal cord of BCP rats and detected the pain behavior of BCP rats.11.Overexpressed lncRNA NONRATT009773.2 and silenced CXCL13 for intervention in the spinal cord of normal rats to observe whether the hyperalgesia caused by lncRNANONRATT009773.2 was reversed.12.Overexpressed lncRNA NONRATT009773,2 and silenced CXCL13 for intervention in the spinal cord of normal rats to observe whether the activation of astrocytes and the up-regulation of inflammatory factors(TNF-α、IL-1β、IL-6)caused by lncRNA NONRATT009773.2 were reversed.Results:1.Bioinformatics analysis showed that among all differentially expressed lncRNAs,lncRNA NONRATT009773.2 was up-regulated most significantly,which may be a key differentially expressed gene in the spinal cord of BCP rats.2.After injection of Walker 256 breast cancer cells into the bone marrow cavity of the tibia,mechanical allodynia and heat hyperalgesia appeared on the 7th day and lasted for more than 21 days.3.RT-PCR results showed that the expression of lncRNA NONRATT009773.2 in the spinal cord of BCP rats increased significantly from 7 days after modeling and lasted for more than 21 days.4.Silencing lncRNA NONRATT009773.2 in the spinal cord of BCP rats reduced hyperalgesia and overexpressing of lncRNA NONRATT009773.2 in the spinal cord of normal rats produced hyperalgesia.5.The results of fluorescence in situ hybridization and immunofluorescence staining showed that lncRNA NONRATT009773.2 were expressed in the cytoplasm of spinal dorsal horn neurons.6.The results of double luciferase reporter gene experiment showed that lncRNA NONRATT009773.2 could directly bind to miR-708-5p.7.The results of double luciferase reporter gene experiment showed that miR-708-5p could directly bind to CXCL13.8.At the animal level,RT-PCR results showed that silencing lncRNA NONRATT009773.2 in the spinal cord of BCP rats caused up-regulation of miR-708-5p expression and down-regulation of CXCL13 expression.Overexpressing lncRNA NONRATT009773.2 in the spinal cord of normal rats caused down-regulation of miR-708-5p expression and up-regulation of CXCL13 expression.9.The results of RT-PCR and ELISA showed that the expression of CXCL13 mRNA and protein in the spinal cord of BCP rats increased significantly from 7 days after modeling and lasted for more than 21 days.10.Silencing the expression of CXCL13 in the spinal cord significantly reduced the hyperalgesia of BCP rats.11.Overexpressed lncRNA NONRATT009773.2 and silenced CXCL13 for intervention in the spinal cord of normal rats.The behavioral results showed that silencing CXCL13 reversed hyperalgesia caused by lncRNA NONRATT009773.2.12.Overexpressed lncRNA NONRATT009773.2 and silenced CXCL13 for intervention in the spinal cord of normal rats.The immunofluorescence results showed that silencing CXCL13 reversed the activation of astrocytes caused by lncRNA NONRATT009773.2.RT-PCR and ELISA results showed that silencing CXCL13 also reversed lncRNA NONRATT009773.2 induced up-regulation of the inflammatory factors(TNF-α,IL-1β,IL-6).Conclusions:The expression of lncRNA NONRATT009773.2 increased in the spinal cord of BCP rats.LncRNA NONRATT009773.2 competitively bound miR-708-5p as a molecular sponge,which relieved the inhibitory effect of miR-708-5p on the target gene CXCL13.Up-regulated expression of CXCL13 caused the activation of astrocytes and the release of inflammatory factors,which produced neuroinflammation,and led to central sensitization and hyperalgesia.