miR-155-5p靶向核因子κB抑制蛋白E在人单核巨噬细胞抗新生隐球菌免疫反应中的机制研究

Mechanistic study on the role of miR-155-5p targeting IKBKE in THP-1 cellsassociated immune response induced by Cryptococcus neoformans

目的分析新生隐球菌(标准株WM148)干预人单核巨噬细胞(THP-1细胞)中miR-155-5p和核因子κB抑制蛋白E(IKBKE)基因的表达变化,验证其靶向关系,并研究其对肿瘤坏死因子细胞(TNF)-α和白细胞介素(IL)-6的调控作用。方法体外培养THP-1细胞,按照数量5∶1将热灭活新生隐球菌加入培养瓶内,分析0h、3h、6h、9h和12h这5个时间点的miR-155-5p和IKBKE的表达倍数变化以及TNF-α和IL-6的表达量变化;双荧光素酶报告系统验证miR-155-5p和IKBKE3'UTR的靶向关系;分别转染miR-155-5pmimics和IKBKEsiRNA,观察在6hmiR-155-5p和IKBKE的表达倍数变化以及TNF-α和IL-6的表达量变化。结果新生隐球菌干预THP-1细胞后,miR-155-5p表达增加,6h达到峰值,随后逐渐下降,而IKBKE在6h表达降低,TNF-α和IL-6的表达量随时间逐渐增加;双荧光素酶报告系统中miR-155-5pmimics作用后IKBKE3'UTR活性下降,将IKBKE3'UTR进行突变后,IKBKE3'UTR活性较野生型增加;分别转染miR-155-5pmimics和IKBKEsiRNA后,在6hTNF-α和IL-6的表达量实验组均较对照组明显增加。结论THP-1细胞中miR-155-5p可通过靶向降解IKBKE信使RNA(mRNA)促进TNF-α和IL-6的表达增加,表明其在THP-1细胞抗新生隐球菌中发挥着促炎作用,可作为将来隐球菌性脑膜炎的潜在治疗靶点。

Objective To analyze expression of miR-155-5p and nuclear factor kappa B inhibitor protein E gene(IKBKE)in human mononuclear macrophages(THP-1 cells)after induction by Cryptococcus neoformans(standard strain WM148),verify the targeting relationship,and study the regulation of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6).Methods THP-1 cells were cultured in vitro.Heat-inactivated Cryptococcus neoformans was added to the culture flask in a ratio of 5:1.The fold change of expression of miR-155-5p and IKBKE and the expression levels of TNF-αand IL-6 were analyzed at 0 h,3 h,6 h,9 h and 12 h.The dual luciferase reporter system verified the targeting relationship between miR-155-5p and IKBKE 3'UTR.miR-155-5p mimics and IKBKE siRNA were transfected,respectively,to observe the fold changes in expression of miR-155-5p and IKBKE as well as the changes in the expression levels of TNF-αand IL-6 at 6 h.Results After addition of Cryptococcus neoformans to THP-1 cells,the expression of miR-155-5p reached peak at 6 h and then decreased gradually,while the expression of IKBKE decreased at 6 h.The expression of TNF-αand IL-6 gradually increased with time.In the dual-luciferase reporter system,the activity of IKBKE 3'UTR decreased after miR-155-5p treatment.After mutation of IKBKE 3'UTR,the activity of IKBKE 3'UTR increased compared with the wild-type.After transfection of miR-155-5p mimics and IKBKE siRNA,the expression levels of TNF-αand IL-6 in the experimental group were significantly higher than those in control group at 6 h.Conclusions miR-155-5p in THP-1 cells can promote the expression of TNF-αand IL-6 by targeting the degradation of IKBKE mRNA,indicating that it plays a proinflammatory role in THP-1 cells against Cryptococcus neoformans.It may be a potential therapeutic target of cryptococcal meningitis.