猪miR-124靶向IQGAP2/CDC42调节吞噬体成熟影响胞内沙门氏菌增殖的机制

IQGAP2/CDC42 are crucial effectors in controlling Salmonella intercellular survival and proliferation in pig, however, the regulation details are not entirely clear. The proposed application intend to carry out research on the mechanisms of miR-124 mediated Salmonella intercellular proliferation, based on the validation of the inhabitation effect of miR-124 on IQGAP2 of pigs. First, the project tries to establish an animal model using piglets by treating with miR-124 liposome nano-vectors and Salmonella bacteria, as well as cell models by over expressing or knocking down miR-124 and IQGAP2; detect the changes of IQGAP2 and downstream CDC42-MAPK signals in Salmonella infected tissues and cells by immunoprecipitation, Real-time PCR and immunofluorescence; elucidate the interaction relationship of miR-124, IQGAP2, and CDC42 by binding site deletion and pull down assay; investigate the maturation process of phagosomes and the number of Salmonella bacteria in tissues and cells by immunofluorescence and other techniques; discover the mechanism of miR-124 intermediated CDC42 inhibition by targeting IQGAP2, and further regulating cell phagocytosis and Salmonella bacteria intracellular survival and proliferation through MAPK pathway. The accomplishment of the project will help to clarify the mechanism of the occurrence and development of salmonella infection, enriching the theoretical knowledge of the molecular mechanism of disease resistance.

IQGAP2/CDC42是调控胞内沙门氏菌增殖的关键，但其调控细节尚不完全清楚。本项目在通过荧光素酶报告实验初步揭示了猪miR-124对IQGAP2抑制作用的基础上，拟开展miR-124介导的胞内沙门氏菌增殖的机制研究。首先建立miR-124脂质体纳米载体处理的沙门氏菌感染仔猪模型、miR-124和IQGAP2超表达或敲低的沙门氏菌感染细胞模型，通过qPCR、免疫组化等技术检测IQGAP2及下游CDC42-MAPK信号的变化，通过位点删除和免疫共沉淀等技术阐明miR-124、IQGAP2和CDC42之间的作用关系，再利用免疫荧光等技术考察组织和细胞中吞噬体成熟、沙门氏菌数量等指标，揭示miR-124抑制IQGAP2并进一步通过CDC42-MAPK途径调节细胞吞噬过程和胞内沙门氏菌增殖的机制。项目的完成将有助于阐明沙门氏菌感染的发生发展机理，丰富猪抗病分子机制的理论知识。

IQGAP2/CDC42是调控胞内沙门氏菌增殖的关键，miR-124在IQGAP2/CDC42调控发挥了重要作用。项目在通过荧光素酶报告实验、EMSA等技术证实了猪miR-124对IQGAP2抑制作用，建立了miR-124 Sponge处理的沙门氏菌感染仔猪模型、miR-124和IQGAP2超表达或敲低的沙门氏菌感染细胞模型，通过qPCR、免疫组化等技术检测发现IQGAP2及下游CDC42-MAPK信号的变化，通过位点删除和免疫共沉淀等技术阐明miR-124、IQGAP2和CDC42之间的作用关系，再利用免疫荧光等技术考察组织和细胞中吞噬体成熟、沙门氏菌数量等指标，结果表明，miR124显著下调CDC42、Rac1等GTPase的活化，抑制沙门氏菌诱导导致的GTPase活化上升，同时，miR-124削弱沙门氏菌引起的pERK2、pJNK1上调作用，表明miR-124经IQGAP2调控CDC42及下游MAPK通路分子。在miR-124处理和CDC42抑制剂处理的巨噬细胞中，Rab7、溶酶体组织蛋白水解酶（CTSD）的转录显著降低，IQGAP2的高表达可促进Rab7和CTSD的转录，miR-124、IQGAP2过表达处理和CDC42抑制剂对感染后期溶酶体相关膜蛋白LAMP-1的转录无显著影响。miR-124和IQGAP2-siRNA处理的巨噬细胞能显著增加细胞内沙门氏菌的数量，尤其是感染后12h，miR-124 sponge显著降低巨噬细胞内沙门氏菌数量。miR-124处理显著降低Lamp+吞噬体。本研究要2个主要发现：（i）miR-124靶向IQGAP2/CDC42介导晚期吞噬体成熟的调控；（ii）miR-124可能通过抑制吞噬溶酶体融合，调控沙门氏菌的胞内增殖。此外，本项目研发了GEREA、Grit软件，为动物的转录因子研究提供了生物信息学工具。项目研究中还构建了表达增强型GFP的沙门氏菌，用于细胞中沙门氏菌的定位与跟踪。本研究发现的miR-124通过靶向IQGAP2/Rho GTPase途径促进猪PBMC中伤寒沙门菌的细胞内增殖的现象，并影响溶酶体—吞噬体融合过程的机理，为建立猪沙门氏菌感染的长期携带状态提供了见解，可为猪沙门氏菌防控药物的开发提供新的思路。