miR-145a-3p通过靶向ATG14调控布鲁氏菌诱导的RAW264.7自噬

【目的】 探究miR-145a-3p对布鲁氏菌诱导的巨噬细胞（RAW264.7）自噬及其对布鲁氏菌胞内生存的影响。【方法】 首先合成自噬相关miRNA miR-145a-3p的模拟物（miR-145a-3p mimics）和抑制剂（miR-145a-3p inhibitor）及对照模拟物（NC mimics），转染至GFP-RFP-RAW264.7细胞中，然后用布鲁氏菌侵染该细胞24 h，通过激光共聚焦显微镜观察miR-145a-3p对自噬的影响；运用TargetScan、miRBase等生物信息学软件预测miR-145a-3p的靶蛋白；通过构建PmirGLO-ATG14-3'UTR和PmirGLO-ATG14-3'UTR-mutation重组质粒，用SacⅠ和KpnⅠ进行双酶切鉴定，并利用双荧光素酶报告系统验证miR-145a-3p与自噬相关基因（autophagy-related gene 14，ATG14）的靶向关系；将RAW264.7细胞培养至60%汇合时，分别转染miR-145a-3p mimics、miR-145a-3p inhibitor和NC mimics，转染7 h后用布鲁氏菌侵染，添加PBS作为未感染对照，培养24 h收集细胞，利用实时荧光定量PCR、Western blotting检测miR-145a-3p对ATG14 mRNA和蛋白表达的调控作用；最后通过布鲁氏菌侵染已转染miR-145a-3p的巨噬细胞，进行菌落计数，验证miR-145a-3p对布鲁氏菌胞内生存的影响。【结果】 miR-145a-3p mimics促进布鲁氏菌诱导的细胞自噬，miR-145a-3p inhibitor抑制布鲁氏菌诱导的细胞自噬；软件预测结果表明，miR-145a-3p靶基因为自噬相关蛋白ATG14-3'UTR；双酶切结果显示，重组质粒PmirGLO-ATG14-3'UTR和PmirGLO-ATG14-3'UTR-mutation构建成功；双荧光素酶报告基因系统验证miR-145a-3p mimics与ATG14-3'UTR互相作用时，与NC mimics组相比，miR-145a-3p mimics组荧光值极显著降低（P<0.01）。与NC mimics组相比，未感染布鲁氏菌时，miR-145a-3p mimics组ATG14 mRNA水平极显著降低（P<0.01）、ATG14蛋白的表达水平显著降低（P<0.05），miR-145a-3p inhibitor组ATG14 mRNA水平极显著上调（P<0.01）；布鲁氏菌感染后，miR-145a-3p mimics+Bru组ATG14 mRNA水平极显著提高（P<0.01），且miR-145a-3p mimics+Bru组ATG14 mRNA的水平显著高于NC mimics+Bru组（P<0.05）。miR-145a-3p mimics促进ATG14蛋白的表达水平。miR-145a-3p的过表达导致布鲁氏菌的胞内生存数量显著降低（P<0.05）。【结论】 miR-145a-3p在布鲁氏菌感染细胞后高表达，miR-145a-3p通过靶向ATG14促进自噬，抑制布鲁氏菌复制。

miR-145a-3p Regulates Brucella Induced Mouse Macrophage RAW264.7 Autophagy by Targeting ATG14

【Objective】 The purpose of this experiment was to investigate the effects of miR-145a-3p on Brucella induced autophagy of macrophages (RAW264.7) and its effect on the intracellular survival of Brucella.【Method】 First, miR-145a-3p mimics, miR-145a-3p inhibitor and NC mimics of autophagy related miRNA miR-145a-3p were synthesized and transfected into GFP-RFP-RAW264.7 macrophages.Then, Brucella infected the cells for 24 h, and the effect of miR-145a-3p on autophagy was observed by laser confocal microscopy.Bioinformatics softwares such as TargetScan and miRBase were used to predict the target protein of mir-145a-3p.The recombinant plasmids PmirGLO-ATG14-3'UTR and PmirGLO-ATG14-3'UTR-mutation were constructed, and were identified by double enzyme digestion with SacⅠ and KpnⅠ, and the targeting relationship between miR-145a-3p and autophagy related gene 14 (ATG14) was verified by double luciferase reporting system.When RAW264.7 cells were cultured to 60% confluence, miR-145a-3p mimics, miR-145a-3p inhibitor and NC mimics were transfected respectively, after transfection for 7 h, they were infected with Brucella, and PBS was added as the uninfected control.The cells were collected after 24 h of culture, and the regulatory effects of miR-145a-3p on ATG14 mRNA and protein expression were detected by Real-time quantitative PCR and Western blotting.Finally, the macrophages transfected with miR-145a-3p were infected by Brucella, and the bacterial colony was counted to verify the effect of miR-145a-3p on the intracellular survival of Brucella.【Result】 miR-145a-3p mimics promoted Brucella induced autophagy, while miR-145a-3p inhibitor inhibited Brucella induced autophagy.The results of software prediction showed that the target of miR-145a-3p was due to the autophagy related protein ATG14-3'UTR.The results of double enzyme digestion showed that the recombinant plasmids PmirGLO-ATG14-3'UTR and PmirGLO-ATG14-3'UTR-mutation were successfully constructed.When the interaction between miR-145a-3p mimics and ATG14-3'UTR was verified by double luciferase reporter gene system, the fluorescence value of miR-145a-3p mimics group was decreased significantly compared with NC mimics group (P<0.01).Compared with the NC mimics group, when not infected with Brucella, in miR-145a-3p mimics group, the level of ATG14 mRNA was extremely significantly decreased (P<0.01) and the level of ATG14 protein was significantly decreased (P<0.05), whlie the level of ATG14 mRNA was significantly increased in miR-145a-3p inhibitor group (P<0.01);After Brucella infection, the level of ATG14 mRNA in miR-145a-3p mimics+Bru group was extremely significantly increased (P<0.01), and the level of ATG14 mRNA in miR-145a-3p mimics+Bru group was significantly higher than that of NC mimics+Bru group (P<0.05).miR-145a-3p mimics promoted the expression level of ATG14 protein.The overexpression of miR-145a-3p decreased the number of intracellular survival of Brucella (P<0.05).【Conclusion】 miR-145a-3p was highly expressed after Brucella infection, and promoted autophagy by targeting ATG14 and inhibited Brucella replication.