HIV派生的MicroRNA99致巨噬细胞焦亡及机制的研究

Impact and mechanism of HIV derived microRNA99 on macrophages pyroptosis

目的探讨HIV派生的MicroRNA99(miRNA99)对巨噬细胞焦亡的影响及机制。方法用佛波酯(PMA)刺激人单核白血病细胞(THP-1)分化成贴壁的巨噬细胞,流式细胞术测定细胞表面CD11b;将鉴定好的细胞分组,分为PBS组(空白对照)、LPS+ATP组(阳性对照)、miRNA99组(实验组);将miRNA99 mimics转染至细胞,采用Western blot(WB)方法测定活化caspase-1蛋白水平;采用ELISA法测定细胞上清液IL-18、IL-1β的水平;将巨噬细胞细胞又分为2组,miRNA99+TLR8 RNAi质粒组实验组、miRNA99+空质粒组,转染细胞,在共聚焦下观察细胞内荧光;采用WB方法检测TLR8蛋白及活化caspase-1蛋白水平;用ELISA法测定TLR8沉默后细胞培养上清液中IL-18、IL-1β的水平。结果经PMA刺激24 h后细胞贴壁生长,伸出伪足,表面CD11b阳性率在99%以上。LPS+ATP组与miRNA99组活化caspase-1蛋白水平、上清液中IL-18、IL-1β的含量明显高于对照组(P<0.05),差异有统计学意义。转染TLR8 RNAi质粒后巨噬细胞内呈现绿色荧光,WB显示实验组TLR8蛋白表达明显减少。在TLR8质粒+miRNA99组,活化caspase-1蛋白水平及上清液中IL-18、IL-1β的含量明显低于空质粒+miRNA99组(P<0.05),差异有统计学意义。结论HIV派生的miRNA99能诱导巨噬细胞发生焦亡,miRNA99诱导焦亡的机制有TLR8途径的参与。

Objective To determine the impact and mechanism of HIV derived microRNA99(miRNA99)on macrophages pyroptosis.Methods THP-1 cells were stimulated by phorbol ester(PMA)and then were cultured and differentiated into sidewall attached macrophages;the morphology and phenotype of CD11b were measured by microscopy and flow cytometry.TLR8 RNAi plasmid was transfected to macrophages and were detected by confocal fluorescence microscopy.The levels of IL-18 and IL-1βreleased by macrophages were measured by ELISA.Western blot(WB)was employed to examine TLR8 and cleaved caspase-1 protein expression in macrophages.Results THP-1 cells that were challenged with PMA(100 ng/ml)for 24 h became smooth and adherent.In addition,the expression of CD11b in macrophages was up to 99%.TLR8 protein expression in macrophages transfected with TLR8 plasmids was significantly lower than that in macrophages transfected with control plasmids.Levels of IL-18 and IL-1βsecreted by macrophages were elevated in LPS+ATP group,miRNA99 group and control plasmid group,but not in control group and TLR8 RNAi plasmid group.Cleaved caspase-1 protein from macrophages of miRNA99 experimental group/LPS+ATP group and control plasmid group was significantly higher than that of control group and TLR8 RNAi plasmid group.Conclusions The present study demonstrates that HIV-derived miRNA99 could induce pyroptosis of macrophages via TLR8-dependent pathway.