果蝇(Drosophila melanogaster)利用天然免疫反应来抵御外源病原菌的入侵,其分子调控机理在进化上非常保守,深入探究果蝇天然免疫调控机制对于人类天然免疫及相关疾病研究具有重要意义。为了发掘参与调控果蝇STING信号依赖的天然免疫反应的新因子,本研究采用双链RNA介导的基因表达沉默技术和双荧光素酶报告系统,在体外S2细胞中对echinus (CG2904)、usp16 (CG4165)、smurf (CG4943)、pellino (CG5212)、usp47 (CG5486)、diap2 (CG8293)、dtraf2 (CG10961)、roquin (CG16807)和usp10 (CG32479)共9个编码泛素连接酶的基因进行双链RNA敲低实验,结果显示CG16807 (roquin)与STING天然免疫信号水平呈现负相关。进一步通过过量表达的方法在S2细胞中过表达roquin能显著抑制STING天然免疫信号。同时,通过李斯特菌感染实验表明,敲低roquin显著提高果蝇感染后抗菌肽的表达,并抑制病原菌的增殖,从而提高果蝇感染后的存活率。本研究证明RNA结合蛋白Roquin是STING依赖的果蝇天然免疫反应的负调控因子,由于果蝇基因和人类基因存在高度相关性,因此本研究为进一步开发人类STING相关的天然免疫疾病治疗方法提供了理论基础。

Drosophila melanogaster utilizes innate immune response to defend against exogenous pathogens. The molecular regulation mechanism of the process is evolutionarily conserved. Research of the regulatory mechanisms of Drosophila innate immunity is greatly significant for understanding the modulation of the human innate immunity and the pathogenesis of related diseases. To explore novel regulators in the STING-dependent innate immune response in Drosophila, we utilized the double-stranded RNA-mediated gene expression silencing technique and the dual-luciferase reporter system in knockdown experiments on 9 genes encoding the ubiquitin ligase such as echinus (CG2904), usp16 (CG4165), smurf (CG4943), pellino (CG5212), usp47 (CG5486), diap2 (CG8293), dtraf2 (CG10961), roquin (CG16807) and usp10 (CG32479) in the S2 cells in vitro. The results suggested a negative correlation between CG16807 (roquin) and the STING signaling pathway. Further studies showed that over-expression of roquin in S2 cells significantly inhibited STING innate immune signaling. Meanwhile, Listeria infection experiments showed that knocking down of roquin markedly elevated the expression levels of anti-microbial peptides and inhibited the proliferation of Listeria, thus increasing the survival rates post pathogenic infection. Taken together, our results suggested that the RNA-binding protein Roquin negatively regulates the STING-dependent innate immune response in Drosophila. In view of the high correlation between Drosophila genes and human genes, this study provides a theoretical basis for further development of treatments for STING-related innate immune diseases in humans.