The Role Of Long Nocoding RNA Cox2 In The Regulation Of Host Anti-listeria Monocytogenes Infection And Its Mechanism

Objective:With the development of genetic testing technique,a large number of studies have indicated that lncRNA could regulate various physiological and pathological functions.In our study,the role of lincRNA-Cox2 and its mechanism in the regulation of host-anti Lm infection were investigated through in vivo and in vitro model.Methods:1.Establishment of Lm-infected RAW264.7 cells model.The levels of IL-1?,IL-6,IL-10 and TNF-? in Lm-infected RAW264.7 cells were detected by qRT-PCR and ELISA.FCM detected the levels of cell surface markers in Lm-infected RAW264.7 cells.qRT-PCR detected the levels of lncRNAs in Lm-infected RAW264.7 cells.2.FISH detected the localization of lincRNA-Cox2 in RAW264.7 cells,the expression of lincRNA-Cox2 was interfered by siRNA.The migration ability of RAW264.7 cells was detected by transwell,the phagocytosis ability of RAW264.7 cells was detected by plate coating,and the proliferation ability of RAW264.7 cells was detected by CCK8.The transcription and secretion levels of inflammatory factors were detected by qRT-PCR and ELISA respectively,the expression of surface markers and cell apoptosis of RAW264.7 cells were detected by FCM,and the protein levels of RAW264.7 cells were detected by Western Blot.3.Western Blot,ELISA and small molecule inhibitor C188-9 were used to detect the mechanism for the changes of biological functions in lincRNA-Cox2 knockdown RAW264.7 cells.The effect of lincRNA-Cox2 knockdown on NF-?B P65 nuclear translocation and the interaction between lincRNA-Cox2 and NF-?B P65 were detected by immunofluorescence and co-localization.4.Lm bacteria liquid was injected through tail vein to establish Lm-infected C57B/6 mice.After infecton for 24 h,blood was collected from eyeballs and the mice were executed.The levels of inflammatory cytokines in peripheral blood plasma were detected by ELISA.The expression level of lincRNA-Cox2 in PBMCs and tissues was detected by qRT-PCR.Colonization of Lm in mouse tissues was detected by Gram staining and PCR.Expression levels of IL-6/JAK3/STAT3 and NF-?B P65 protein in mouse tissues were detected by Western Blot.Result:1.The transcription and secretion levels of inflammatory cytokines IL-1?,IL-6 and TNF-? were increased in Lm-infection RAW264.7 cells,while the transcription and secretion levels of IL-10 have no significant changes.FCM results show that Lm infection could upregulate the surface markers of CD80 and MHC II in RAW264.7 cells,while CD86 expression has no significant changes.qRT-PCR results showed that Lm infection resulted in differential expression of lncRNAs in RAW264.7 cells,and the levels of lincRNA-Cox2 was increased with the extension of infection time,the expression was highest at 6 h.2.FISH result showed that lincRNA-Cox2 was located in the cytoplasm and nucleus of RAW264.7 cells,mainly in the cytoplasm.However,lincRNA-Cox2 was mainly located in the nucleus in Lm-infected RAW264.7 cells.LincRNA-Cox2 siRNA-1009 can effectively inhibit the expression of lincRNA-Cox2 Down-regulation expression of lincRNA-Cox2 in RAW264.7 cells can inhibit the cell migration ability and promote the phagocytosis ability in RAW264.7 cells.However,results showed that inhibiting of lincRNA-Cox2 expression had no effect on cell proliferation,cell cycle and cell surface markers in RAW264.7 cells.Results of qRT-PCR and ELISA showed that lincRNA-Cox2 knockdown inhibited the secretion of pro-inflammatory cytokines(IL-1?,IL-6 and TNF-?)in Lm-infected RAW264.7 cells.FCM and Western Blot results indicated that lincRNA-Cox2 knockdown inhibited the apoptosis rate and cleaved Caspase-3 induced by Lm infection in RAW264.7 cells.3.Western Blot and ELISA results showed that down-regulation of lincRNA-Cox2 in Lm-infected RAW264.7 cells have no effect on p38MAPK and ERK1/2,while it could inhibit IL-6/JAK3/STAT3 pathway leading to decreased migration ability and enhanced phagocytosis in RAW264.7 cells infected by Lm Immunofluorescence assay showed that down-regulation of lincRNA-Cox2 inhibited the phosphorylation and nucleus translocation of NF-?B P65,lincRNA-Cox2 and NF-?B P65 had the same intracellular localization in RAW264.7 cells.4.The expression of pro-inflammatory cytokines in peripheral blood plasma were increased in Lm-infected C57B/6 mice.Gram staining and PCR results showed that Lm was remained in PBMCs and could colonize in liver,spleen and brain tissues Results of qRT-PCR showed that the levels of lincRNA-Cox2 in PBMCs and tissues of mice were significantly increased,and the most obvious elevation was in liver Western Blot showed that Lm infection could increase the expression levels and phosphorylation of IL-6/JAK3/STAT3 and NF-?B P65 in tissues of mice,the most obvious elevation was in liver.Conclusion:Lm infection leads to increasing expression of lincRNA-Cox2 in RAW264.7 cells,which can affect the inflammatory response and regulate the migration and phagocytosis of RAW264.7 cells through adjusting with IL-6/JAK3/STAT3 signal pathway.Meanwhile,lincRNA-Cox2 interacts with NF-?B P65 to regulate the NF-kB P65 activation and nucleus translocation.The results were also verified in mice.