The lifestyle transition of fungi,defined as switching from taking organic material as nutrients to pathogens,is a fundamental phenomenon in nature.However,the mechanisms of such transition remain largely unknown.Here...The lifestyle transition of fungi,defined as switching from taking organic material as nutrients to pathogens,is a fundamental phenomenon in nature.However,the mechanisms of such transition remain largely unknown.Here we show microRNA-like RNAs(milRNAs)play a key role in fungal lifestyle transition for the first time.We identified milRNAs by small RNA sequencing in Arthrobotrys oligospora,a known nematode-trapping fungus.Among them,7 highly expressed milRNAs were confirmed by northern-blot analysis.Knocking out two milRNAs significantly decreased A.oligospora’s ability to switch lifestyles.We further identified that two of these milRNAs were associated with argonaute protein QDE-2 by RNA-immunoprecipitation(RIP)analysis.Three of the predicted target genes of milRNAs were found in immunoprecipitation(IP)products of QDE-2.Disruption of argonaute gene qde-2 also led to serious defects in lifestyle transition.Interestingly,knocking out individual milRNAs or qde-2 lead to diverse responses under different conditions,and qde-2 itself may be targeted by the milRNAs.Collectively,it indicates the lifestyle transition of fungi is mediated by milRNAs through RNA interference(RNAi)machinery,revealing the wide existence of miRNAs in fungi kingdom and providing new insights into understanding the adaptation of fungi from scavengers to predators and the mechanisms underlying fungal infections.展开更多
目的应用氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的小鼠巨噬细胞株制备动脉粥样硬化模型,观察微小RNA-1270(microRNA-1270,miRNA-1270)是否经血管生成素样蛋白7(angiopoietin-like protein 7,ANGPTL7)/p38途径...目的应用氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的小鼠巨噬细胞株制备动脉粥样硬化模型,观察微小RNA-1270(microRNA-1270,miRNA-1270)是否经血管生成素样蛋白7(angiopoietin-like protein 7,ANGPTL7)/p38途径干预巨噬细胞炎症及血脂代谢。方法培养小鼠单核巨噬细胞(RAW264.7),并加入ox-LDL构建动脉粥样硬化模型。根据干预条件不同分为空白组、ox-LDL组、ANGPTL7组、p38蛋白抑制组、p65蛋白抑制组、miR-1270模拟物组、miR-1270模拟物阴性对照组、miR-1270抑制物组和miR-1270抑制物阴性对照组。采用实时定量PCR法检测mRNA表达水平,采用Western blotting法检测蛋白质表达水平,采用油红染色法检测脂质蓄积。结果ox-LDL暴露的巨噬细胞中含红色脂肪微粒的巨噬细胞数量增加,ANGPTL7高表达,p38高表达,白介素-6(interleukin-6,IL-6)高表达,白介素-10(interleukin-10,IL-10)低表达,且ANGPTL7与p38及脂质蓄积呈正相关(P<0.01)。与ox-LDL组相比,ANGPTL7组ANGPTL7、p38及IL-6蛋白相对表达量增加,IL-10蛋白相对表达量减少,含红色脂肪微粒的巨噬细胞数量增加(P<0.01)。与ANGPTL7组相比,p38蛋白抑制组ANGPTL7和p38蛋白相对表达量无明显差异,IL-6蛋白相对表达量减少,IL-10蛋白相对表达量增加,含红色脂肪微粒的巨噬细胞数量减少(P<0.01)。与ANGPTL7组相比,p65蛋白抑制组各项指标表达量、含红色脂肪微粒的巨噬细胞数量差异无统计学意义(P>0.05)。与空白组相比,ox-LDL组miR-1270基因相对表达量减少,ANGPTL7基因及蛋白相对表达量增加,miR-1270与ANGPTL7基因相对表达量呈负相关(P<0.01)。与ox-LDL组相比,miR-1270模拟物组miR-1270基因相对表达量增加,ANGPTL7基因及蛋白相对表达量减少,p38及IL-6蛋白相对表达量减少,IL-10蛋白相对表达量增加,含红色脂肪微粒的巨噬细胞数量减少(P<0.01);miR-1270抑制物组miR-1270基因相对表达量减少,ANGPTL7基因及蛋白相对表达量增加,p38及IL-6蛋白相对表达量增加,IL-10蛋白相对表达量减少,含红色脂肪微粒的巨噬细胞数量增加(P<0.01);与ox-LDL组相比,阴性对照物组各项指标表达量无明显差异,含红色脂肪微粒的巨噬细胞数量差异无统计学意义(P>0.05)。结论在ox-LDL暴露的巨噬细胞模型中,ANGPTL7促进巨噬细胞内的炎症和脂质蓄积必经p38途径实现,是促进动脉粥样硬化的发生发展新机制。miR-1270作为保护性因子,可靶向抑制ANGPTL7基因转录表达,经p38途径减轻巨噬细胞炎症及脂质蓄积,可逆调控动脉粥样硬化发生发展,是动脉粥样硬化潜在的早期筛查靶点。展开更多
基金This work was supported by the National Basic Research Program of China(2013CB127500)the National Natural Science Foundation of China(31160021,31270131 and U1502262)+1 种基金sponsored by the Nanjing University of Posts and Telecommunications Scientific Foundation(NUPTSF)(NY218140)a grant(2018KF003)from YNCUB.We thank BGI-Shenzhen who contributed to the small RNA sequencing projects.We thank H.Yin for comments and discussion.
文摘The lifestyle transition of fungi,defined as switching from taking organic material as nutrients to pathogens,is a fundamental phenomenon in nature.However,the mechanisms of such transition remain largely unknown.Here we show microRNA-like RNAs(milRNAs)play a key role in fungal lifestyle transition for the first time.We identified milRNAs by small RNA sequencing in Arthrobotrys oligospora,a known nematode-trapping fungus.Among them,7 highly expressed milRNAs were confirmed by northern-blot analysis.Knocking out two milRNAs significantly decreased A.oligospora’s ability to switch lifestyles.We further identified that two of these milRNAs were associated with argonaute protein QDE-2 by RNA-immunoprecipitation(RIP)analysis.Three of the predicted target genes of milRNAs were found in immunoprecipitation(IP)products of QDE-2.Disruption of argonaute gene qde-2 also led to serious defects in lifestyle transition.Interestingly,knocking out individual milRNAs or qde-2 lead to diverse responses under different conditions,and qde-2 itself may be targeted by the milRNAs.Collectively,it indicates the lifestyle transition of fungi is mediated by milRNAs through RNA interference(RNAi)machinery,revealing the wide existence of miRNAs in fungi kingdom and providing new insights into understanding the adaptation of fungi from scavengers to predators and the mechanisms underlying fungal infections.
