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

doi:10.16431/j.cnki.1671-7236.2022.03.034

Abstract

Abstract: 【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.

Key words: Brucella; miRNA; autophagy; autophagy associated protein ATG14; intracellular survival

CLC Number:

S852.61⁺4

WANG Yueli, SHAO Zhiran, YI Jihai, WANG Yong, WANG Zhen, CHEN Chuangfu. miR-145a-3p Regulates Brucella Induced Mouse Macrophage RAW264.7 Autophagy by Targeting ATG14[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1117-1125.

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

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