egr-miR-71过表达对绵羊PBMCs蛋白组的差异表达分析

为了探究细粒棘球绦虫(Echinococcus granulosus)来源的miR-71(egr-miR-71)对绵羊外周血单核细胞(PBMCs)蛋白组的影响。本研究使用密度梯度离心法分离得到绵羊PBMCs,并使用电穿孔法将egr-miR-71转染至绵羊PBMCs,用qPCR检测转染效率。基于iTRAQ方法,对比分析egr-miR-71过表达对绵羊PBMCs蛋白表达的影响,通过qPCR和Western-blotting对骨髓源性生长因子(MYDGF)转录水平和翻译水平进行验证。结果显示,绵羊PBMCs转染egr-miR-71模拟物后,miR-71的相对表达量极显著上调(P<0.01)。利用iTRAQ共鉴定出7642个蛋白质,其中157个呈差异表达,与对照组相比,68个蛋白显著上调,89个蛋白显著下调。下调的蛋白主要包括巨噬细胞迁移抑制因子(MIF)、MYDGF、酰基辅酶A结合蛋白(ACBP)和碳酸酐酶(CAs)等。这些差异蛋白主要参与丝裂原活化蛋白激酶(MAPK)、转化生长因子-β(TGF-β)等信号通路。qPCR和Western-blotting结果显示,与对照组相比,模拟物转染组中MYDGF转录和翻译水平均显著下调(P<0.05)。通过对转染egr-miR-71后绵羊PBMCs的蛋白进行组学分析,初步了解了egr-miR-71对绵羊PMBCs表达蛋白的调控,鉴定出的差异蛋白可能在细粒棘球蚴与宿主互作中发挥重要作用。这为深入研究细粒棘球绦虫的感染机制提供了思路,也为新型抗包虫病药物的研发提供了理论依据。

Effects of egr-miR-71 over-expression on the proteome of sheep PBMCs

The study aims to investigate the effects of Echinococcus granulosus miR-71(egr-miR-71)overexpression on the proteome of sheep peripheral blood mononuclear cells(PBMCs).PBMCs were obtained via density gradient centrifugation,and then egr-miR-71 was transfected into PBMCs by electroporation.The transfection efficiency was validated by q PCR.Furthermore,the effects of overexpressed egr-miR-71 on sheep PBMCs proteome were analyzed via isobaric rags for relative and absolute quantitation(iTRAQ).The m RNA and protein levels of MYDGF were identified by q PCR and Western blotting,respectively.The result showed that the relative expression level of egr-miR-71 was significantly up-regulated after transfection(P<0.01).A total of 7642 proteins were identified by iTRAQ,157 of which were differentially expressed.Among them,68 proteins were up-regulated and 89 were down-regulated.The down-regulated proteins mainly included macrophage migration inhibitory factor(MIF),bone marrow-derived growth factor(MYDGF),acyl-Co A binding protein(ACBP),and carbonic anhydrase(CAs).Moreover,the differentiallyexpressed proteins were mainly involved in the regulation of signaling pathways,such as mitogen-activated protein kinase(MAPK)and transforming growth factor-beta(TGF-beta).The results showed that the m RNA and protein levels of MYDGF were significantly down-regulated in the mimics-transfection group compared with the control group(P<0.01).The current results will provide a preliminary understanding for the regulation of host PMBC-expressed proteins.The differentially-expressed proteins might play important roles in the interactions between E.granulosus and the host,which provides ideas for further exploring the infectious mechanisms,and also lays the foundation for the development of new anti-echinococcosis drugs.