Role of microRNAs in Regulating Gene Expression

我们正在利用生物信息学/进化保守分析来确定microRNAs(MiRNAs)是否可以靶向干扰素-伽马mRNA。为了解决这种可能性，我们在人类自然杀伤细胞系NK92中产生了稳定的DICER基因的小干扰RNA(SiRNA)。DICER是一种对整个miRNA加工至关重要的酶，因此，如果miRNAs参与基因表达，那么DICER水平的降低可能会改变干扰素-伽马基因的表达。结果表明，在DICER基因敲除的NK92转染体中，干扰素-γ的表达可能会因特定的刺激而改变，从而暗示miRNAs参与了干扰素-γ的表达调控。此外，我们在干扰素-γ3‘非翻译区发现了一个进化上保守的miRNA结合位点，初步数据表明该位点有助于调节干扰素-γ的表达。这种miRNA与干扰素-γmRNA的结合可能会导致RNA的构象改变，从而由于mRNA的稳定而导致干扰素-伽马的表达增加。这种稳定性将代表一种新的模型，说明miRNA在调节干扰素-伽马基因表达中的作用。除了干扰素-伽马基因，我们正在与玛丽·卡林顿博士的实验室合作，研究miRNAs在调节HLSA-C基因表达方面的可能作用。初步结果已在该基因的3‘非翻译区发现了一个miRNA结合位点，并在该区域发现了多态。目前的工作集中在表征这个miRNA结合位点在人类白细胞抗原-C细胞表面表达中的作用，以及SNPs对该基因表达的影响。作为整个项目的一部分，我们还在寻找可能通过改变干扰素-g基因稳定性来影响基因表达的干扰素-γ基因的多态。

We are utilizing bioinformatics/evolutionary conservation analysis to determine if microRNAs (miRNAs) may target the interferon-gamma mRNA. To address this possibility, we have generated a stable small interfering RNA (siRNA) transfectant of the dicer gene in the human natural killer cell line NK92. Dicer is an enzyme critical for overall miRNA processing and thus decreased dicer levels may alter interferon-gamma gene expression if miRNAs are involved in gene expression. Results indicate that interferon-gamma expression may be altered in response to specific stimuli in the dicer knockdown NK92 transfectant, thus implicating miRNAs in the regulation of interferon-gamma expression. Furthermore, we have identified an evolutionarily conserved miRNA binding site in the interferon-gamma 3' untranslated region and preliminary data indicates that this site contributes to regulating interferon-gamma expression. Binding of this miRNA to the IFN-gamma mRNA may result in an altered conformation of the RNA, resulting in increased IFN-gamma expression due to a stabilization of the mRNA. Such a stabilization would represent a new model for the role of a miRNA in regulating IFN-gamma gene expression. In addition to the IFN-gamma gene, we are investigating the possible role of miRNAs in regulating HLSA-C gene expression, in collboration with the laboratory of Dr. Mary Carrington. Preliminary results have identified a miRNA binding site in the 3' untranslated region of the gene and polymorphisms have been identified in this region. current efforts are focused on characterizing the role of this miRNA binding site in HLA-C cell surface expression and the impact of the SNPs on this gene expression. As part of this overall project, we are also searching for polymorphisms in the IFN-gamma gene that may effect gene expression by altering IFN-g mRNA stability.