METHODS: A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis in vitro. The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay.
RESULTS: The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively.
CONCLUSIONS: ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells.
方法:人类单核细胞系,THP-1细胞,被刺激以诱导体外脓毒症模型。ATP2B1-AS1,miR-23a-3p,通过实时定量聚合酶链反应评估TLR4。通过流式细胞术探讨ATP2B1-AS1在细胞凋亡和炎症中的作用。蛋白质印迹分析和酶联免疫吸附血清学测定。ATP2B1-AS1和miR-23a-3p之间的结合位点,和miR-23a-3p和TLR4之间的预测BiBiServ和RNA相互作用的百科全书(ENCORI)在线网站,分别,并通过荧光素酶测定证实。
结果:在脂多糖(LPS)处理的THP-1细胞中,ATP2B1-AS1的水平升高。LPS增加细胞凋亡率,促凋亡因子的相对蛋白表达,和相对信使RNA(mRNA)水平和促炎细胞因子的浓度,但降低了抗凋亡蛋白的相对表达和相对mRNA水平以及抗炎因子的浓度。通过将shATP2B1-AS1转染到THP-1细胞中可以逆转所有这些改变。此外,ATP2B1-AS1直接结合miR-23a-3p并负调节miR-23a-3p的水平。同时,TLR4被miR-23a-3p直接靶向,以及分别由miR-23a-3p和ATP2B1-AS1负和正调节。
结论:ATP2B1-AS1通过调节LPS处理的THP-1细胞中miR-23a-3p/TLR4轴加重细胞凋亡和炎症反应。