MiR-1696 regulates NETs formation in broilers by inhibiting respiratory burst and the PI3K/AKT and PKC/MAPK pathways via targeting Gpx3

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.

图1 微小RNA-1696（miRNA-1696）靶向肉鸡中性粒细胞中的谷胱甘肽过氧化物酶3（Gpx3）。
A. 检测不同转染浓度（模拟物mimics与抑制剂inhibitors）下中性粒细胞内miRNA-1696的mRNA水平。
B. 检测转染模拟物或抑制剂的中性粒细胞内Gpx3的mRNA与蛋白水平。
C. 采用双荧光素酶实验验证miRNA-1696与Gpx3的靶向关系。

图2 谷胱甘肽过氧化物酶3（Gpx3）参与肉鸡中性粒细胞胞外陷阱（NETs）的形成。
A. 经佛波醇酯（PMA）刺激2小时后，采用扫描电子显微镜（SEM）观察NETs的形成情况。
B. 经PMA刺激2小时后，使用SYTOX Green染料对中性粒细胞与NETs进行染色，采用荧光显微镜（FM）观察NETs的形成情况。
C. 在Gpx3敲低（knock-down）组与对照组中，检测Gpx3、髓过氧化物酶（MPO）与蛋白激酶C（PKC）的mRNA与蛋白水平。

图3 微小RNA-1696（miRNA-1696）参与肉鸡中性粒细胞胞外陷阱（NETs）的形成。
A. 经PMA刺激2小时后，采用SEM观察NETs形成情况。
B. 经PMA刺激2小时后，使用SYTOX Green染料对中性粒细胞与NETs染色，采用FM观察NETs形成情况。
C. 在Gpx3敲低组与对照组中，检测Gpx3、MPO与PKC的mRNA与蛋白水平。
数据以平均值±标准差（n=5）表示，采用Tukey检验，*P < 0.05。
D. 经PMA刺激2小时后，使用SYTOX Green染料对中性粒细胞与NETs染色，采用FM观察NETs形成情况。
E. 在Gpx3敲低组与对照组中，检测Gpx3、MPO与PKC的mRNA与蛋白水平。

图4 谷胱甘肽过氧化物酶3（Gpx3）与miRNA-1696介导的氧化还原状态调控。
A-B. 采用二氯二氢荧光素-双乙酸酯（DCFH-DA，绿色荧光，5 mM）进行免疫荧光实验检测细胞内活性氧（ROS）的生成情况，所用中性粒细胞转染了siRNA-Gpx3。
C-D. 采用DCFH-DA进行免疫荧光实验检测细胞内ROS生成情况，所用中性粒细胞分别经Gpx3过表达质粒、Gpx3过表达质粒与miRNA-1696模拟物共转染，以及仅转染miRNA-1696模拟物处理。

图5 Gpx3敲低的肉鸡中性粒细胞中磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）与丝裂原活化蛋白激酶（MAPKs）通路的mRNA与蛋白水平。
对转染siRNA-Gpx3的中性粒细胞与对照组中性粒细胞，采用实时荧光定量聚合酶链式反应（RT-PCR）检测PI3K、AKT、Erk、JNK与P38的mRNA水平；采用蛋白质免疫印迹（Western blot）检测上述细胞的蛋白水平。

图6 miRNA-1696通过靶向Gpx3调控中性粒细胞中PI3K/AKT与MAPKs通路的mRNA与蛋白水平。
对分别转染Gpx3过表达质粒、miRNA-1696模拟物，以及共转染Gpx3过表达质粒与miRNA-1696模拟物的中性粒细胞，采用RT-PCR检测PI3K、AKT、Erk、JNK与P38的mRNA水平；采用Western blot检测上述细胞的蛋白水平。
数据以平均值±标准差（n=5）表示，无相同字母的柱形组间存在显著差异（p < 0.05），采用Tukey检验，*P < 0.05。