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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 diseases and tumors. MicroRNA is a negative regulator of molecules. The aim of this study is to explore the regulatory role of microRNA on Fas gene. Bioinformatics analysis of microRNAs that may regulate the expression of Fas gene and its effect on Fas gene expression by polymerase chain reaction (RT-PCR) and flow cytometry. Site-directed mutagenesis and double fluorescent reporter systems were used to verify the direct regulation of Fas mRNA by microRNAs. Finally, the effect of microRNA on Fas-mediated apoptosis was detected by flow cytometry. As a result, bioinformatics analysis revealed that the let-7 / miR-98 family can regulate Fas gene expression. Overexpression of miR-98 can reduce the expression of Fas gene protein and mRNA levels. On the contrary, inhibition of miR-98 can increase the expression of Fas gene. Double fluorescent reporter system and site-directed mutagenesis experiments show that let-7 / miR-98 family can directly regulate Fas gene 3’UTR. Apoptosis detection showed that overexpression of miR-98 can inhibit Fas-mediated apoptosis, and inhibition of miR-98 can increase Fas-mediated apoptosis. This indicates that the let-7 / miR-98 family can directly regulate Fas gene expression and affect Fas-mediated apoptosis, and may be a new drug that interferes with the related diseases of Fas involvement.