MiR-548d-3p调节过氧化氢刺激下人圆锥角膜成纤维细胞IL-1β的表达

MiR-548d-3p Regulates IL-1βExpression in Human Keratoconic Corneal Fibroblasts under Oxidative Stress

氧化应激和炎性因子与圆锥角膜发病密切相关。MiRNAs作为重要的基因表达调控因子参与了角膜的生理病理过程。为了探讨MiRNAs是否通过靶向IL-1β调控圆锥角膜成纤维细胞中与基质重塑相关基因的表达,通过生物信息学软件预测可能与IL-1β靶定的MiRNAs(miR-21-5p,miR-24-3p,miR-26b-3p,miR-27b-5p,miR-181d-5p,miR-383-3p,MiR-548d-3p,miR-4700-3p),对体外培养的人圆锥角膜成纤维细胞添加过氧化氢处理0,6,12,24 h,采用实时荧光定量PCR检测上述MiRNAs及IL-1β基因的表达,并通过双荧光素酶报告分析实验确定MiR-548d-3p与IL-1β之间存在靶定关系。将MiR-548d-3p的模拟物转染至人圆锥角膜成纤维细胞内,发现在氧化应激条件下,MiR-548d-3p的模拟物可显著下调IL-1β的基因表达,并使MMP-1和MMP-3表达下降、Collagen I-α2表达升高。提示氧化应激条件下,人圆锥角膜成纤维细胞MiR-548d-3p可通过下调IL-1β表达促进角膜基质合成,减少降解,进而减缓圆锥角膜病变进程,为圆锥角膜患者治疗提供潜在靶点。

Oxidative stress and inflammatory factors are closely related to keratoconus.As an important gene expression regulator,MiRNAs participate in the physiological and pathological process of keratoconus.In this study,in order to investigate whether MiRNAs can regulate the expression of matrix remodeling-related genes in keratoconus fibroblasts by targeting IL-1β,potential MiRNAs(miR-21-5p,miR-24-3p,miR-26b-3p,miR-27b-5p,miR-181d-5p,miR-383-3p,MiR-548d-3p,miR-4700-3p)that may be related to IL-1βtargeting were predicated by using bioinformatics methods.Hydrogen peroxide was added to human keratoconic corneal fibroblasts in vitro.quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of MiRNAs and IL-1βmRNA at 0,6,12,and 24 h.IL-1βwas found to be a target of MiR-548d-3p by using a dual luciferase reporter assay.After transfection of MiR-548d-3p mimics into human keratoconus fibroblasts,it was found that under oxidative stress,MiR-548d-3p mimics significantly down-regulated the gene expression of IL-1β,decreased the expression of MMP-1 and MMP-3,and increased the expression of collagenI-α2.These results indicate that MiR-548d-3p may promote corneal matrix synthesis and attenuate matrix degradation by down-regulating the expression of IL-1β,which retards keratoconus progression under oxidative stress.This might provide a new potential target for keratoconus treatment.