LncRNA NR003508通过海绵吸附miR-483-3p并靶向MLKL调控BCG感染小鼠巨噬细胞坏死

LncRNA NR003508 Regulates BCG-infected Mouse Macrophages Necrosis by the Sponge Adsorption of miR-483-3p and Targeting MLKL

旨在探讨LncRNA NR_003508在牛分枝杆菌卡介苗(Bacillus Calmette-Guérin, BCG)感染中对小鼠巨噬细胞坏死的调控作用。本研究利用小干扰RNA技术干扰小鼠巨噬细胞系RAW264.7中LncRNA NR_003508的表达,并结合BCG感染,采用Western blot、免疫荧光和流式细胞术等技术检测坏死相关调控因子混合谱系激酶结构域样蛋白(MLKL)的表达水平和细胞坏死率,以及通过生物信息学和双荧光素酶报告基因技术分别预测和验证LncRNA NR_003508与miR-483-3p的靶向结合。结果表明,BCG感染巨噬细胞后,LncRNA NR_003508表达水平显著上调(P<0.01);转染siRNA-LncRNA NR_003508后,MLKL蛋白表达显著下调(P<0.01),同时巨噬细胞坏死率也显著降低(P<0.05);LncRNA NR_003508可以与miR-483-3p特异性结合并发挥海绵吸附作用,并且干扰LncRNA NR_003508可显著上调miR-483-3p的表达(P<0.01),而过表达miR-483-3p显著抑制MLKL的表达(P<0.01),同时抑制巨噬细胞坏死。以上研究结果表明,LncRNA NR_003508通过海绵吸附miR-483-3p并靶向MLKL的表达来促进BCG诱导的巨噬细胞坏死。

To investigate the function of LncRNA NR_003508 in regulating Bacillus Calmette-Guérin(BCG)-induced necrosis of mouse macrophages, small interfering RNA for LncRNA NR_003508 were transfected into RAW264.7 cells alone or combined with BCG infection. Western blot, immunofluorescence and flow cytometry were used to detect the expression level of MLKL(mixed lineage kinase domain-like protein) and cell necrosis rate;The target binding of LncRNA NR_003508 and miR-483-3 p was predicted and verified by bioinformatics and dual-luciferase report assay. The results showed that after BCG infection of macrophages, the expression level of LncRNA NR_003508 was significantly increased(P<0.01);After transfection of siRNA-LncRNA NR_003508, the protein expression of MLKL was significantly down-regulated(P<0.01), and the rate of macrophage necrosis was also significantly reduced(P<0.05). LncRNA NR_003508 adsorbed miR-483-3 p through sponge effect, and interference of LncRNA NR_003508 significantly up-regulated the expression of miR-483-3 p(P<0.01), while overexpression of miR-483-3 p significantly inhibited the expression of MLKL(P<0.01) and macrophage necrosis. Our results indicated that LncRNA NR_003508 promoted BCG-induced macrophages necrosis by sponging miR-483-3 p and targeting the expression of MLKL.