摘要
目的 分析环状RNA（circRNA）在脂多糖（LPS）诱导小鼠腹腔巨噬细胞炎症反应中的表达差异,初步探讨mmu_circ_0000790（以下简称为circ790）对LPS诱导小鼠腹腔巨噬细胞分泌促炎性细胞因子的影响.方法 提取C57BL/6雌性小鼠腹腔巨噬细胞,用1 mg/L LPS刺激巨噬细胞,12 h后收集细胞及上清液,采用酶联免疫吸附试验（ELISA）检测上清液中炎症因子IL-1β、IL-6和TNF-α的含量;利用circRNA芯片检测LPS诱导组（n=3）和空白对照组（n=3）细胞中circRNA表达谱的变化.采用实时荧光定量PCR（RT-PCR）对部分差异表达的circRNA进行验证,并以circ790为研究对象,检测LPS诱导巨噬细胞炎症反应中circ790的动态变化.用小干扰RNA诱导小鼠腹腔巨噬细胞中circ790沉默,检测炎症因子IL-1β、IL-6和TNF-α的表达变化;运用生物信息学方法预测circ790相关的microRNA.结果 与空白对照组相比,LPS诱导巨噬细胞12 h后上清液中IL-1β、IL-6、TNF-α含量明显升高（P〈0.01）,证实巨噬细胞炎症反应模型构建成功.芯片筛查结果提示有34个circRNA的表达在LPS诱导组和对照组之间的差异有统计学意义（P〈0.05）,其中12个circRNA上调,22个circRNA下调.RT-PCR验证结果和芯片结果一致.LPS刺激巨噬细胞3、6、12 h后,circ790表达水平较空白对照组上调了（1.94＆#177;0.15）、（3.18＆#177;0.13）和（4.21＆#177;0.22）倍（P〈0.05）.在转染si-circ790后,IL-6的分泌水平明显降低（P〈0.05）,但对IL-1β和TNF-α的水平没有明显影响（P〉0.05）.生物信息学提示circ790可能通过与microRNA发生相互作用从而调控巨噬细胞炎症反应.结论 LPS诱导小鼠腹腔巨噬细胞炎症反应后,circRNA表达谱发生显著变化,circ790可能参与调控了巨噬细胞分泌IL-6.
Objective To analyze the expression profile of circular RNA （circRNA） in LPS-treated murine peritoneal macrophages （MPMs） and to investigate the effects of mmu_circ_0000790 （circ790） on the secretion of pro-inflammatory cytokines in MPMs following LPS stimulation.Methods MPMs were isolated from C57BL/6 male mice and then stimulated with （LPS treatment group） or without （blank control group） 1 mg/L of LPS for 12 hours.Supernatants of cell culture and cell pellets were collected from each group.Enzyme linked immunosorbent assay （ELISA） was used to measure the changes in the secretion of IL-1β,IL-6 and TNF-α in the supernatants.Expression profile of circRNA in macrophages from the two groups （n=3） was analyze by circRNA microarray.Some differentially expressed circRNAs were validated by real-time PCR （RT-PCR）.Moreover,RT-PCR was also performed to detect the expression of circ790 in MPMs after LPS stimulation.Small interfering RNA （siRNA） oligo specific for circ790 was designed and synthesized.Then the synthesized siRNA oligo and normal control （NC） oligo were transfected into MPMs by LipofectamineTM2000.The transfected MPMs were treated with LPS.ELISA was used to detect the levels of IL-1β,IL-6 and TNF-α in the supernatants.Arraystar＆#39;s home-made microRNA target prediction software was used to predict circ790/microRNA.Results The concentrations of IL-1β,IL-6 and TNF-α in the LPS treatment group were significantly higher than those in the blank control group （P〈0.01）.These results indicated that the inflammatory model of MPMs was successfully constructed.Statistical differences in 34 differentially expressed circRNAs were found between the two groups （P〈0.05）.Among them,12 circRNAs were up-regulated and the other 22 circRNAs were down-regulated in the LPS treatment group.RT-PCR results were generally consistent with the microarray data.The expression of circ790 was increased by （1.94＆#177;0.15）,（3.18＆#177;0.13） and （4.21＆#177;0.22） folds
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2017年第6期405-411,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（81560001）
江西省科技支撑计划项目（20151BBG70208）
关键词
脂多糖
巨噬细胞
炎症反应
Circular RNA
LPS
Macrophage
Inflammatory response
