沉默HDAC6对嗜肺军团菌干扰RAW264.7自噬作用的影响

Effects of silencing HDAC6 on the autophagy of RAW264.7 cells infected by Legionella pneumophila

目的以嗜肺军团菌感染HDAC6基因沉默的RAW264.7细胞为研究对象,检测自噬流及自噬相关因子表达水平的变化,探究HDAC6在嗜肺军团菌干扰巨噬细胞自噬中的作用机制。方法利用RNAi技术介导基因沉默,构建shRNAHDAC6细胞,Western blot验证沉默效果。用嗜肺军团菌活菌和灭活菌[感染复数(MOI)=50]分别感染RAW264.7、sh-NC、shRNA-HDAC6细胞6、12、24 h。应用自噬双标质粒pmCherry-C1-EGFP-LC3B检测巨噬细胞自噬流的变化;透射电镜观察嗜肺军团菌感染24 h后各组细胞内的自噬小体和自噬溶酶体;实时荧光定量PCR及Western blot法检测AMBRA1、ATG9B、P62、Beclin1、α-tubulin、LC3B的表达水平。结果与RAW264.7对照组相比,shRNA-HDAC6细胞的HDAC6蛋白表达水平显著降低(P<0.05);自噬双标质粒检测自噬流结果显示,嗜肺军团菌活菌及灭活菌感染RAW264.7细胞,自噬流均减弱,沉默HDAC6后嗜肺军团菌感染巨噬细胞自噬流增加。透射电镜可观察到嗜肺军团菌感染巨噬细胞24 h各组的自噬小体及自噬溶酶体。实时荧光定量PCR及Western blot检测结果显示,与RAW264.7对照组相比,嗜肺军团菌感染RAW264.7细胞后,ATG9B、α-tubulin、P62的表达水平均明显增加(P<0.05),LC3B、Beclin1和AMBRA1的表达水平均明显下调(P<0.05);与shRNA-HDAC6对照组相比,嗜肺军团菌感染shRNA-HDAC6细胞后,α-tubulin的表达水平明显下降(P<0.05)。结论嗜肺军团菌可抑制巨噬细胞的自噬,沉默HDAC6后嗜肺军团菌对巨噬细胞自噬的抑制作用减弱,自噬流增加,提示嗜肺军团菌可能通过与HDAC6相互作用而影响微管蛋白乙酰化及细胞应激等,进而干扰巨噬细胞的自噬。

This study was performed to investigate the effects of silencing HDAC6 on the autophagy of RAW264.7 cells infected by Legionella pneumophila(LP).The expression of HDAC6 in RAW264.7 cells was silenced by RNA interference.The experimental groups includes RAW264.7,sh-NC and shRNA-HDAC6,which incubated with activated or inactivated LP[MOI=50]for 6,12,24 h.The pmCherry-GFP-LC3B tandem fluorescent protein was used to monitor the autophagy flux;transmission electron microscopy(TEM)was used to observe the autophagosomes and autolysosomes in each group of cells infected by LP for 24 h.Apart from that,the mRNA and protein expression levels of Beclin1,AMBRA1,ATG9B,P62,α-tubulin and LC3B were detected by RT-qPCR and Western blotting.Data showed that the protein expression of HDAC6 was significantly decreased in shRNA-HDAC6,compared with RAW264.7 and sh-NC group(P<0.05).When LP infected RAW264.7 cells,theautophagy flux was clearly inhibited;when LP infectedsh RNA-HDAC6,the autophagy flux was increased,asmonitored by pm Cherry-C1-EGFP-LC3 B.RT-q PCR and Western blot analysis revealed that when RAW264.7 and sh-NC cells were infected by LP,the expression ofATG9 B,α-tubulin and P62 increased(P<0.05),but the expression of LC3 B,Beclin1 and AMBRA1 decreased(P<0.05).When sh RNA-HDAC6 cells were infected by LP,the expression ofα-tubulin decreased,as compared withthe sh RNA-HDAC6 group(P<0.05).In conclusion,LP can inhibit autophagy flux in macrophages,and silencingHDAC6 can cut down this inhibition,suggesting that LP may affect the acetylation of tubulin and cellular stress toinfluence the autophagy by interacting with HDAC6.