猪miR-124靶向IQGAP2调节巨噬细胞内沙门氏菌的增殖

Porcine miR-124 target IQGAP2 and regulating intracellular Salmonella proliferation in macrophage cells

【目的】明确猪miR-124与其靶基因IQGAP2间的表达调控关系,以及miR-124表达水平与猪巨噬细胞内沙门氏菌数量的关联,为揭示沙门氏菌在感染细胞内存活与增殖的机制提供理论依据。【方法】通过荧光素酶报告基因系统验证miR-124与IQGAP2基因的作用位点;再以GM-CSF诱导的猪巨噬细胞和鼠伤寒沙门氏菌(ATCC 14028)为试验材料,通过实时荧光定量PCR和流式细胞术测定沙门氏菌感染猪巨噬细胞中miR-124和IQGAP2基因的表达及巨噬细胞内沙门氏菌的增殖情况。【结果】miR-124结合位点野生型载体转染的荧光报告信号显著低于miR-124结合位点突变载体(P<0.05,下同),但共转染anti-miR-124序列后能显著增强miR-124结合位点野生型载体的荧光报告信号。经沙门氏菌感染后,猪巨噬细胞中的miR-124表达被激活,感染12、24和48 h后的相对表达量均显著高于沙门氏菌感染前(0 h),而IQGAP2基因表达水平呈显著下调趋势;在沙门氏菌感染猪巨噬细胞内,miR-124表达水平与IQGAP2基因表达水平呈明显负相关(r=-0.92)。miR-124高表达组细胞内的沙门氏菌数量显著高于正常巨噬细胞,但miR-124敲低表达组细胞内的沙门氏菌数量显著低于正常巨噬细胞;IQGAP2基因敲低表达组细胞内的沙门氏菌数量显著高于正常巨噬细胞;此外,miR-124高表达+IQGAP2基因敲低表达组细胞内的沙门氏菌数量与IQGAP2基因敲低表达处理组相比无显著差异(P>0.05),但显著高于miR-124高表达组细胞。【结论】沙门氏菌感染猪巨噬细胞中的miR-124表达水平与IQGAP2基因表达水平及胞内沙门氏菌数量呈负相关,即沙门氏菌可通过上调miR-124表达靶向抑制IQGAP2基因表达,从而调节其在猪巨噬细胞内的增殖。

【Objective】To explore the regulation relationship between miR-124 and its target gene IQGAP2,study the relationship between the expression level of miR-124 and the number of Salmonella in pig macrophages,so as to provide a theoretical basis for revealing the mechanism of survival and proliferation of Salmonella in infected cells.【Method】The binding sitebetween miR-124 and IQGAP2 was verified by luciferase reporter gene system assay.Then,the GM-CSF induced macrophages and Salmonella typhimurium(ATCC 14028)were used as research materials.The expression of miR-124 and IQGAP2 gene and the proliferation of Salmonella in macrophages were determined by real-time quantitative PCR and flow cytometry.【Result】The fluorescence signal of wild-type vector transfected by miR-124 binding site was significantly lower than that of miR-124 binding site mutant vector(P<0.05,the same below),and the co-transfection of antimiR-124 sequence could significantly enhance the fluorescence signal of wild-type vector.In the process of Salmonella infection,the expression of miR-124 was activated,the relative expression levels at 12,24 and 48 h after infection were significantly higher than those before infection(0 h).The expression of IQGAP2 was significantly down-regulated.In Salmonella infected macrophages,the expression levels of miR-124 were negatively correlated with IQGAP2(r=-0.92).The Salmonella counts in miR-124 high expression group were significantly higher than the control.The Salmonella counts in miR-124 knock down group were significantly lower than the control macrophages.The Salmonella counts in miR-124 knock-down group were significantly higher than the control macrophagesgroup.The numberof Salmonella counts in miR-124 high expression+IQGAP2 gene knockdown expression group were not significantly different compared to the IQGAP2 knockdown group(P>0.05),but significantly higher than the miR-124 high expression group cells.【Conclusion】The expression levels of miR-124 and IQGAP2 in Salmonella infected porcine macrophages are negatively correlated with intercellular Salmonella counts.Salmonella inhibits IQGAP2 gene expression by up-regulating miR-124 expression targeting,thereby regulating its proliferation in porcine macrophages.