MiR-1696 regulates NETs formation in broilers by inhibiting respiratory burst and the PI3K/AKT and PKC/MAPK pathways via targeting Gpx3
Description
Fig.1 MiRNA-1696 targets Gpx3 in broiler neutrophils. A. The mRNA levels of miR-1696 were detected in neutrophils with different transfection concentration (mimics and inhibitors). B. The mRNA and protein levels of Gpx3 were detected in neutrophils with mimics or inhibitors. C. Dual luciferase verified the target relationship between miR-1696 and Gpx3. Fig.2 Gpx3 participates in NETs formation in broiler. A. After stimulating by PMA for 2 hours, NETs formation was observed by using SEM. B. After stimulating by PMA for 2 hours, the neutrophils and NETs are stained by SYTOX green, and NETs formation was observed by using FM. C. The mRNA and protein levels of Gpx3, MPO, and PKC were detected in Gpx3 knock-down group and control group. Fig.3 MiR-1696 participates in NETs formation in broiler. A. After stimulating by PMA for 2 hours, NETs formation was observed by using SEM. B. After stimulating by PMA for 2 hours, the neutrophils and NETs are stained by SYTOX green, and NETs formation was observed by using FM. C. The mRNA and protein levels of Gpx3, MPO, and PKC were detected in Gpx3 knock-down group and control group. Date were presented as mean ± SD (n=5). *P < 0.05 by Tukey test. D. After stimulating by PMA for 2 hours, the neutrophils and NETs are stained by SYTOX green, and NETs formation was observed by using FM. E. The mRNA and protein levels of Gpx3, MPO, and PKC were detected in Gpx3 knock-down group and control group. Fig.4 Gpx3 and miR-1696-mediated modulation of redox states. A-B. ROS generation was performed by immunofluorescence using DCFH-DA (green fluorescence, 5 mM) in cells. Neutrophils were treated with transfected by siRNA-Gpx3. C-D. ROS generation was performed by immunofluorescence using DCFH-DA (green fluorescence, 5 mM) in cells. Neutrophils were treated with transfected by Gpx3 plasmid, both Gpx3 plasmid and miR-1696 mimics, and miRA-1696 mimics, respectively. Fig.5 The mRNA and protein levels of PI3K/AKT and MAPKs pathways in Gpx3 knock-down neutrophils. The mRNA levels of PI3K, AKT, Erk, JNK and P38 were detected by RT-PCR in neutrophils transfected with siRNA-Gpx3 and control neutrophils. The protein levels were detected by western blot in neutrophils transfected with siRNA-Gpx3 and control neutrophils. Fig.6 MiR-1696 regulates the mRNA and protein levels of PI3K/AKT and MAPKs pathways neutrophils by targeting Gpx3. The mRNA levels of PI3K, AKT, Erk, JNK and P38 were detected by RT-PCR in neutrophils transfected with Gpx3- plasmid, miR-1696 mimics and Gpx3-plasmid, miR-1696 mimics, respectively. The protein levels were detected by western blot in neutrophils transfected with Gpx3- plasmid, miR-1696 mimics and Gpx3-plasmid, miR-1696 mimics, respectively. Date were presented as mean ± SD (n=5). Bars that do not share the same letters are significantly different (p <0.05) from each other, and *P < 0.05 by Tukey test.