Effects of IRF3 knockdown on the nuclear expression of Irak1bp1 protein in LPS-stimulated Raw 264.7 cells
10.3969/j.issn.1674-8115.2018.06.001?
- VernacularTitle:敲减干扰素调节因子3对脂多糖刺激Raw 264.7细胞核内Irak1bp1表达的影响
- Author:
Zhi-Li TAN
1
;
Ying-Ying WANG
;
Huan ZHONG
;
Yu HE
;
Qing-Qing SHI
;
Xue YANG
;
Guo-Rong XU
;
Liang-Ming LIU
Author Information
1. 上海交通大学附属第一人民医院松江分院感染科
- Keywords:
interferon regulator factor 3;
short hairpin RNA;
interleukin-1 receptor associated kinase 1 binding protein 1 (Irak1bp1);
lipopolysaccharide (LPS)
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2018;38(6):589-593
- CountryChina
- Language:Chinese
-
Abstract:
Objective·To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods?·?Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results?·?The titer of adenovirus was 2.2×1011PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion?·?Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.
