miRNA-155靶向SOCS5对无乳链球菌诱导罗非鱼脑星形胶质细胞炎症的影响

Effect of MiRNA-155-Targeted SOCS5 Gene on Inflammatory Response of Brain Astrocytes of Oreochromis niloticus Induced by Streptococcus agalactiae

【目的】探究尼罗罗非鱼(Oreochromis niloticus)微小RNA-155(miR-155)和SOCS5(Suppressor of cytokine signaling 5)的靶向关系,并研究其对无乳链球菌诱导的尼罗罗非鱼脑星形胶质细胞炎症反应的影响。【方法】通过实时荧光定量PCR(qRT-PCR)检测感染无乳链球菌后尼罗罗非鱼脑星形胶质细胞中miR-155、SOCS5及炎症因子表达变化规律,生物信息学方法预测miR-155与SOCS5的靶向关系,双荧光素酶报告基因实验进行靶基因验证,qRT-PCR方法分析过表达和敲降miR-155对SOCS5基因表达的影响和miR-155对SOCS5的调控机制。【结果】无乳链球菌诱导尼罗罗非鱼脑星形胶质细胞中miR-155表达量极显著上升(P<0.001),4 h达到最高值。SOCS5表达量极显著上升(P<0.01),6 h达到最高值。促炎症因子IL-1β、TNF-α表达显著升高(P<0.0001),抑炎症因子IL-10、TGF-β表达显著下调(P<0.05)。构建尼罗罗非鱼SOCS53′UTR野生型和突变型荧光素酶报告载体。双荧光素酶实验结果表明miR-155极显著抑制(P<0.0001)SOCS5野生型质粒表达。突变靶位点后,miR-155对SOCS5的抑制作用极显著降低(P<0.0001),抑制效果减弱53.7%。过表达miR-155能显著抑制(P<0.001)SOCS5表达,而抑制miR-155后SOCS5表达显著上升(P<0.05)。转染miR-155抑制物6 h和12 h后,miR-155表达量显著下调(P<0.01),12 h表达量最低,而SOCS5表达量显著上调(P<0.05),在12 h达到最高值。【结论】miR-155通过靶向负调控SOCS5的表达参与无乳链球菌诱导的尼罗罗非鱼脑星形胶质细胞炎症反应,通过下调SOCS5表达影响炎症变化。

【Objective】To explore the targeting relationship between microRNA-155(miR-155)and SOCS5(Suppressor of cytokine signaling 5)in Nile tilapia(Oreochromis niloticus),and to study its effect on astrocyte inflammation induced by Streptococcus agalactis in O.niloticus.【Method】The expression of miR-155,SOCS5 and inflammatory factors in brain astrocytes of O.niloticus infected with S.agalactis were detected by real-time fluorescence quantitative PCR(qRT-PCR).The targeting relationship between miR-155 and SOCS5 was predicted by bioinformatics method,and the targeted gene was verified by double luciferase reporter gene experiment.QRT-PCR method was used to analyze the effect of overexpression and knockdown of miR-155 on SOCS5 gene expression and the regulatory mechanism of miR-155 on SOCS5.【Result】The expression of miR-155 in brain astrocytes of O.niloticus induced by S.agalactis increased significantly(P<0.001),and reached the highest level at 4 h.The expression of SOCS5 increased significantly(P<0.01),and reached the highest at 6 h.The expression of pro-inflammatory cytokines IL-1βand TNF-αincreased significantly(P<0.0001),while the expression of anti-inflammatory factors IL-10 and TGF-βdecreased significantly(P<0.0001).The wild type and mutant luciferase reporter vectors of O.niloticus SOCS53'UTR were successfully constructed.The results of double luciferase experiment showed that miR-155 significantly inhibited(P<0.0001)the expression of SOCS5 wild type plasmid.After mutating the target site,the inhibitory effect of miR-155 on SOCS5 decreased significantly(P<0.0001)by 53.7%.Overexpression of miR-155 could significantly inhibit the expression of SOCS5(P<0.001).Compared with NC group,the expression of SOCS5 increased significantly after inhibition of miR-155(P<0.001).The expression of miR-155 was significantly down-regulated at 6 h and 12 h after transfection of miR-155 inhibitor,and the expression was the lowest at 12 h,while the expression of SOCS5 was significantly up-regulated(P<0.05),and reached the peak at 12 h.【Conclusion】The negative regulation of SOCS5 expression targeted by miR-155 participates in the inflammatory response of astrocytes of O.niloticus induced by S.agalactis,and affects the occurrence and development of inflammation by down-regulating the expression of SOCS5.