日本血吸虫mir-125b靶向调控宿主CD276的鉴定研究

摘要

To validate Schistosoma japonicum (S.japonicum) mir-125b targeting murine CD276 and analyze the expression of CD276 in different tissues of mice infected with S.japonicum,we predicted in our previous study that Schistosomajaponicum mir-125b might target murine CD276.Here,we used PCR to amplify the putatively interacted regions of murine CD276 and cloned the amplified fragment into the downstream of a Gaussia luciferase vector (pGluc-Basic).Then,the recombinant plasmids mir-125b mimics and anti-sense mir-125b were transfected into HEK293 T cells and the luciferase activities were measured by using a dual luciferase reporter system.In addition,the expression of CD276 in vitro cultured murine macrophages was also determined in quantitative real-time PCR upon mir-125b mimics transfection.Finally,the expression of CD276 in different tissues of mice infected with S.japonicum was also determined in quantitative real-time PCR.The putatively interacted region of CD276 was obtained and the corresponding recombinant plasmid was successfully constructed.A significant down-regulation of luciferase reporter activity was observed upon transfection ofmir-125b mimics into HEK293T cells (P ＜ 0.05).Transfection ofmir-125b mimics into murine macrophages led to the down-regulation of CD276.In addition,the expression of CD276 in lymphocytes,livers,and spleens of mice infected with S.japonicum was also down-regulated (P ＜ 0.05).Our results indicated that S.japonicum mir-125b directly target murine CD2 76 during schistosome infection,suggesting that mir-125b might play an important regulatory role in parasite-host interactions.%通过荧光素酶实验验证宿主CD276为日本血吸虫mir-125b的靶基因,并分析感染血吸虫后宿主CD27基因在不同组织中的表达情况.利用PCR扩增mir-125b与CD2 76互作的核酸序列,并将目的片段克隆至表达荧光素酶报告基因质粒的下游.将重组质粒转染到HEK293T细胞内,检测荧光素酶报告基因表达.进一步利用荧光定量PCR技术检测小鼠巨噬细胞在转染血吸虫mir-125b mimic后,靶基因CD276的表达情况,同时检测感染血吸虫不同时间段、小鼠不同组织中CD276的表达情况.荧光素酶实验表明转染mir-125b mimic和重组质粒后细胞的荧光素酶报告基因的表达极显著降低(P＜0.01),提示mir-125b可与CD276相关区域互作,抑制报告基因的表达;血吸虫mir-125b mimic转染小鼠巨噬细胞后导致其宿主CD276基因的表达降低,进一步提示宿主CD276可能为血吸虫mir-125b靶基因;荧光定量PCR分析表明感染日本血吸虫后的不同感染时期,小鼠淋巴细胞、肝脏及脾脏中的CD276的表达水平显著低于对照组(P＜0.01),进一步表明虫源性mir-125b在体内影响宿主CD276基因的表达.日本血吸虫mir-125b可能靶向调控宿主CD276基因的表达,在虫体与宿主互作中发挥重要调控功能.