let-7/miR-98家族对Fas基因的调节作用

The regulation of Fas expression by let-7/miR-98 family

Fas在自身免疫病及肿瘤中发挥重要的作用。微小RNA是一种起负调控作用的分子。本研究旨在探索微小RNA对Fas基因的调节作用。生物信息学分析可能调节Fas基因表达的微小RNA,并通过聚合酶链反应(RT-PCR)和流式细胞术检测其对Fas基因表达的影响。利用定点突变和双荧光报告基因系统来验证微小RNA对Fas基因的直接调节作用。最后通过流式细胞术检测微小RNA对Fas介导的细胞凋亡的影响。结果,生物信息学分析发现let-7/miR-98家族可以调节Fas基因的表达。过表达miR-98可以降低Fas基因蛋白和mRNA水平的表达,反之,抑制miR-98可以增加Fas基因的表达。双荧光报告基因系统及定点突变实验显示:let-7/miR-98家族可直接调节Fas基因3'UTR。细胞凋亡检测显示:过表达miR-98可抑制Fas介导的细胞凋亡,抑制miR-98可以增加Fas介导的细胞凋亡。这说明let-7/miR-98家族可以直接调节Fas基因的表达并影响Fas介导的细胞凋亡,并可能成为干预Fas参与的相关疾病的一种新药物。

Fas plays an important role in autoimmune and neoplastic disease,and microRNA acts as a negative regulator that modulates target gene expression.This study was to investigate the microRNA which can regulate the expression and function of Fas.The microRNA which can regulate Fas expression was analyzed by bioinformatics.Real-time PCR and flow cytometry were used to test Fas mRNA and protein expression levels,respectively.Site-directed gene mutagenesis and reporter gene assay were performed to identify microRNA exerting their regulation on Fas 3＇UTR directly.Apoptosis was assessed by annexin-V staining using flow cytometry.The experimental results showed that let-7/miR-98 family was predicted to regulate Fas by bioinformatics.Over-expression of miR-98 significantly reduced the expression of Fas at both protein and messenger RNA levels, whereas reduction of miR-98 expression conversely increased Fas at both levels.Furthermore,let-7/miR-98 could target Fas 3＇UTR directly.More importantly,over-expression of miR-98 decreased cellular＇ sensitivity to Fas-induced apoptosis, and vise versa.These findings indicate that let-7/miR-98 down-regulate Fas expression and decrease cellular sensitivity to Fasinduced apoptosis.And let-7/miR-98 may provide a potential novel strategy for therapeutic intervention of Fas-related diseases.