2023, 43(2): 1-10. doi: 10.3969/j.issn.1673-9159.2023.02.001
关键词： 微小核糖核酸155（miR-155） , SOCS5 , 炎症反应 , 无乳链球菌 , 罗非鱼脑星形胶质细胞
Keywords： microRNA-155 (miR-155) , SOCS5 , inflammatory reaction , Streptococcus agalactiae , brain astrocytes of Oreochromis niloticus
【目的】探究尼罗罗非鱼（Oreochromis niloticus）微小RNA-155（miR-155）和SOCS5（Suppressor of cytokinesignaling 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 < 0.000 1），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SOCS5 3′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 < 0.000 1） 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 < 0.000 1），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-1β and TNF-α increased significantly (P < 0.000 1), while the expression of anti-inflammatory factors IL-10 and TGF-β decreased significantly (P < 0.000 1). The wild type and mutant luciferase reporter vectors of O.niloticus SOCS5 3'UTR were successfully constructed. The results of double luciferase experiment showed that miR-155 significantly inhibited (P < 0.000 1) the expression of SOCS5 wild type plasmid. After mutating the target site, the inhibitory effect of miR-155 on SOCS5 decreased significantly (P < 0.000 1) 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.
| [1] | 黄德生.罗非鱼链球菌病的诊治[J].畜牧兽医科技信息, 2021(4):200. |
| [2] | 张行,李新圃,杨峰,等.无乳链球菌研究进展[J].中国兽医学报, 2020, 40(4):864-872. |
| [3] | 何洁.无乳链球菌对罗非鱼头肾损伤的病理学研究[D].雅安:四川农业大学, 2018. |
| [4] | 祝璟琳,杨弘.鱼源无乳链球菌致病机理研究进展[J].广东海洋大学学报, 2013, 33(6):92-96. |
| [5] | LI Y Y, WANG J, ZHANG C P, et al. Research progress of microRNAs and related factors in the pathogenesis of lichen planus[J]. International Journal of Dermatology and Venereology, 2020, 3(3):171-175. |
| [6] | 郑荣立,姜玉杰,王欣.非霍奇金淋巴瘤耐药相关microRNA研究进展[J].中国实验血液学杂志, 2014, 22(5):1490-1494. |
| [7] | PASCA S, JURJ A, PETRUSHEV B, et al. microRNA-155 implication in M1 polarization and the impact in inflammatory diseases[J]. Frontiers in Immunology, 2020, 11:625. |
| [8] | 李江,胡维新. microRNA-155研究进展[J].生命科学, 2010, 22(2):139-142. |
| [9] | WANG B, GAN Z, CAI S H, et al. Comprehensive identification and profiling of Nile tilapia (Oreochromis niloticus) microRNAs response to Streptococcus agalactiae infection through high-throughput sequencing[J]. Fish&Shellfish Immunology, 2016, 54:93-106. |
| [10] | 常晶,姚战非,张铎,等.基于miR-155/SOCS1轴研究复方甘草酸苷治疗特应性皮炎小鼠的作用机制[J].中国免疫学杂志, 2020, 36(13):1583-1588. |
| [11] | 闵敏. microRNA-155通过调节转录因子FOXO3a在溃疡性结肠炎中的作用[D].厦门:中国人民解放军军医进修学院, 2012. |
| [12] | 赵家洁,游晶,李静,等. SOCS的免疫调节机制及其在慢性肝病中的作用[J].中国免疫学杂志, 2022, 38(17):2140-2146. |
| [13] | 刘青娟.炎症在糖尿病肾病发病中的作用及细胞因子信号转导抑制因子SOCS-1、SOCS-3的影响[D].石家庄:河北医科大学, 2010. |
| [14] | 夏倩. SOCS3和SOCS5在RSV感染支气管上皮细胞的TH1/TH2免疫失衡中的表达[D].长沙:中南大学, 2010. |
| [15] | 陈景行,朱述阳.罗格列酮对哮喘小鼠气道炎症和肺组织细胞因子信号抑制因子3和5 mRNA表达的影响[J].郑州大学学报(医学版), 2011, 46(2):198-201. |
| [16] | LI Y, HUANG Y, CAI J, et al. Establishment of an astrocytelike cell line from the brain of tilapia (Oreochromis niloticus) for virus pathogenesis and a vitro model of the blood-brain barrier[J]. Journal of Fish Diseases, 2022, 45(10):1451-1462. |
| [17] | WANG B, JIAN J C, LU Y S, et al. Complete genome sequence of Streptococcus agalactiae ZQ0910, a pathogen causing meningoencephalitis in the GIFT strain of Nile tilapia (Oreochromis niloticus)[J]. Journal of Bacteriology, 2012, 194(18):5132-5133. |
| [18] | 廖宁馨. microRNA-155靶向SOCS1基因在新生隐球菌诱导巨噬细胞炎症反应中的调控作用[D].上海:第二军医大学, 2016. |
| [19] | KAYANSAMRUAJ P, PIRARAT N, HIRONO I, et al. Increasing of temperature induces pathogenicity of Streptococcus agalactiae and the up-regulation of inflammatory related genes in infected Nile tilapia (Oreochromis niloticus)[J]. Veterinary Microbiology, 2014, 172(1/2):265-271. |
| [20] | 刘华子. 80例新生儿无乳链球菌脑膜炎和大肠埃希菌脑膜炎的临床分析[D].杭州:浙江大学, 2020. |
| [21] | 罗素.鱼源无乳链球菌小鼠血脑屏障开放评估模型的建立及cas9基因功能分析[D].南京:南京农业大学, 2016. |
| [22] | 于柯瑶,张克明,黄越,等. Dectin-1在宿主抗隐球菌感染的机制与研究进展[J].广西医科大学学报, 2023, 40(1):1-5. |
| [23] | 崔静雯.尼罗罗非鱼无乳链球菌脑膜炎病理损伤的动态研究及无乳链球菌透明质酸酶多克隆抗体制备[D].雅安:四川农业大学, 2015. |
| [24] | 胡古月,杨静,黄瑞.细菌性脑膜炎患者脑脊液、血清lncRNA NEAT1水平与病情及血脑屏障破坏程度相关性分析[J].中国病原生物学杂志, 2023, 18(2):207-210. |
| [25] | 陈秀慧,张晓沁,杨舜芳,等.白细胞介素-10、CD138、促血管生成素-2在子宫内膜炎中表达水平及相关性[J].临床军医杂志, 2023, 51(2):210-213. |
| [26] | 赵瑾,张晓丽,王能,等.针刺联合补肝益肾强膝方对膝骨性关节炎患者(肝肾亏虚型) TGF-β1、COX-2、TNF-α及骨代谢指标的影响[J].中国老年学杂志, 2023, 43(1):59-63. |
| [27] | 颜晓龙.鮸鱼IL-1β的miRNA调控研究[D].舟山:浙江海洋大学, 2019. |
| [28] | 郭兰兰,邢正,张玉寒,等.肌源性microRNA-133的研究进展[J].生命的化学, 2022, 42(4):727-736. |
| [29] | WU X Y, PAIS E M A, LI L, et al. microRNA-155:a novel armamentarium against inflammatory diseases[J]. Inflammation, 2017, 40(2):708-716. |
| [30] | TAN L, JIANG W, LU A, et al. miR-155 aggravates liver ischemia/reperfusion injury by suppressing SOCS1 in mice[J]. Transplantation Proceedings, 2018, 50(10):3831-3839. |
| [31] | 程爽. miRNA靶基因预测及其功能识别算法研究[D].哈尔滨:哈尔滨工业大学, 2016. |
| [32] | 石瑜,李振东,李可,等. miR-155-5p靶向调控SOCS5调节JAK2/STAT3信号通路促进大鼠脑缺血-再灌注损伤[J].脑与神经疾病杂志, 2021, 29(9):572-577. |
| [33] | FUJIMOTO M, NAKA T. Development of SOCS gene therapy for intractable cancers:the discovery and therapeutic application of SOCS[J]. Annals of Oncology, 2016, 27:vii14. |
| [34] | WANG B, WANGKAHART E, SECOMBES C J, et al. Insights into the evolution of the suppressors of cytokine signaling (SOCS) gene family in vertebrates[J]. Molecular Biology and Evolution, 2019, 36(2):393-411. |
| [35] | 王运九,孙健,李玲霞,等. miR-190通过调控细胞因子信号转导抑制因子5促进肝细胞癌发生的机制[J].新医学, 2021, 52(9):703-708. |
| [36] | 罗定安. SOCS5在缺氧诱导肝细胞癌侵袭转移中的作用及其潜在机制的研究[D].青岛:青岛大学, 2022. |
| [37] | BI H Q, LI Z H, ZHANG H. Long noncoding RNA HAND2-AS1 reduced the viability of hepatocellular carcinoma via targeting microRNA-300/SOCS5 axis[J]. Hepatobiliary&Pancreatic Diseases International, 2020, 19(6):567-574. |
| [38] | 张琴. miR-301a靶向SOCS5调节JAK-STAT3信号通路参与胰腺癌侵袭转移[D].武汉:湖北中医药大学, 2018. |
| [39] | 张贺.二氢杨梅素通过抑制小胶质细胞中NF-κB和JAK2/STAT3信号通路来减轻脂多糖诱导的炎症反应[D].南京:南京大学, 2017. |
| [40] | 李婷.外泌体源性miRNA-155靶向调控巨噬细胞极化影响溃疡性结肠炎的机制研究[D].乌鲁木齐:新疆医科大学, 2022. |
| [41] | 梁音,张剑,王新卫,等. miRNA-155靶向Wnt5a调控转录激活子3信号通路在支气管哮喘发生发展中的作用[J].医学研究生学报, 2020, 33(9):931-936. |
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关键词: 微小核糖核酸155（miR-155） SOCS5 炎症反应 无乳链球菌 罗非鱼脑星形胶质细胞
2023, 43(2): 1-10.
