Effect of Notch1, 2, 3 genes silencing on Notch and nuclear factor-κB signaling pathway of macrophages derived from patients with coronary artery disease

Abstract

Objective: To investigate the effects of Notch1, 2, 3 genes silencing by siRNA on Notch signaling pathway (Delta-like 4(DLL4), Jagged 1(JAG1)) and nuclear factor-κB (NF-κB) signaling pathway (IκBα, P52) of macrophages derived from patients with coronary artery disease (CAD), thus to explore the potential genetic treatment perspectives for CAD. Methods: Peripheral blood mononuclear cells of CAD patients were isolated by density gradient centrifugation and transformed by phorbol-12-myristate-13-acetate (PMA) to macrophages. Macrophages were then transfected with Notch1-small interference RNA (siRNA, Notch1-siRNA group), Notch2-siRNA (Notch2-siRNA group), Notch3-siRNA (Notch3-siRNA group), negative control siRNA (NC group) and none siRNA (control group) respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were applied to assess the mRNA and protein expression levels of DLL4, JAG1, IκBα and p52, respectively. Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity. Subcellular distributions of NF-κB/p52 were detected through immunofluorescence. Results: (1) The mRNA and protein expressions of DLL4, JAG1 and p52 in Notch1-siRNA group, Notch2-siRNA group and Notch3-siRNA group were significantly downregulated, while the mRNA and protein expression of IκBα was significantly upregulated compared with NC group and control group(P<0.05 or 0.01). The mRNA and protein expressions of DLL4, JAG1 and p52 in Notch1-siRNA group were significantly downregulated, while the mRNA and protein expression of IκBα was significantly upregulated compared with Notch2-siRNA group and Notch3-siRNA group(P<0.05 or 0.01). The mRNA and protein expressions of DLL4, JAG1, IκBα and p52 were similar between NC group and control group (all P>0.05). (2) The binding activity of NF-κB DNA was significantly lower in Notch1-siRNA group (613±57), Notch2-siRNA group (1 169±85) and Notch3-siRNA group (1 454±90) compared with control group (2 643±115) and NC group (2 407±100) (all P<0.01), which was also significantly lower in Notch1-siRNA group compared to Notch2-siRNA group and Notch3-siRNA group (P<0.01); was significantly lower in Notch2-siRNA group compared with Notch3-siRNA group (P<0.01) and was similar between control group and NC group (P>0.05). (3) The fluorescence intensity of NF-κB/p52 was significantly lower both in the nucleus and cytoplasm in Notch1-siRNA group, Notch2-siRNA group and Notch3-siRNA group compared with NC group and control group (all P<0.01), and the decrease was more obviously in the nucleus than in cytoplasm in Notch1-siRNA group, Notch2-siRNA group and Notch3-siRNA group (P<0.05 or 0.01). The fluorescence intensity of NF-κB/p52 was similar between control group and NC group (P>0.05). Conclusion: There is a positive regulation between Notch and NF-κB pathway in macrophages derived from CAD patients, the regulation power on NF-κB signaling pathway of Notch1 is stronger than that of Notch2 and Notch 3.