METHODS: MRC-5 cells were incubated with lipopolysaccharide (LPS) for 12 h to establish the in vitro cellular model for infantile pneumonia. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of circ_0044411, miR-141-3p (micoRNA-141-3p) and CCL16 (CC motif chemokine ligand 16). Cell viability and proliferation was assessed by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-2\'-deoxyuridine (EdU) assay. The levels of inflammatory factors IL-1β, IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay kits. Cell apoptosis and caspase-3 activity were detected by flow cytometry analysis and caspase-3 activity assay kit. The target interaction was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay.
RESULTS: Circ_0044411 was highly repressed in the serum of infantile pneumonia patients and LPS-induced MRC-5 cells. Circ_0044411 could promote the cell viability and proliferation, inhibit inflammatory response and apoptosis in LPS-induced MRC-5 cells. Circ_0044411 could serve as a sponge of miR-141-3p, and miR-141-3p could reverse the function of circ_0044411 on LPS-induced MRC-5 cell injury. In addition, miR-141-3p could target CCL16, and miR-141-3p could protect MRC-5 cells from LPS-induced cell injury by targeting CCL16. Furthermore, circ_0044411 sponged miR-141-3p to positively regulate CCL16 expression.
CONCLUSIONS: Circ_0044411 knockdown promoted cell viability and proliferation, inhibited inflammatory response and apoptosis by regulating miR-141-3p/CCL16 axis, indicating that circ_0044411 might be a potential therapeutic target for IP.
方法:将MRC-5细胞与脂多糖(LPS)孵育12h,建立婴儿肺炎的体外细胞模型。逆转录-定量聚合酶链反应(RT-qPCR)用于检测circ_0044411、miR-141-3p(micoRNA-141-3p)和CCL16(CC基序趋化因子配体16)的水平。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)测定和5-乙炔基-2'-脱氧尿苷(EdU)测定来评估细胞活力和增殖。炎症因子IL-1β水平,采用酶联免疫吸附测定试剂盒测定IL-6和TNF-α。流式细胞术和caspase-3活性检测试剂盒检测细胞凋亡和caspase-3活性。通过双荧光素酶报告基因测定证实了靶相互作用,RNA免疫沉淀测定和RNA下拉测定。
结果:Circ_0044411在小儿肺炎患者血清和LPS诱导的MRC-5细胞中被高度抑制。Circ_0044411能促进细胞活力和增殖,抑制LPS诱导的MRC-5细胞的炎症反应和凋亡。Circ_0044411可以作为miR-141-3p的海绵,miR-141-3p可以逆转circ_0044411对LPS诱导的MRC-5细胞损伤的作用。此外,miR-141-3p可以靶向CCL16,miR-141-3p可以通过靶向CCL16保护MRC-5细胞免受LPS诱导的细胞损伤。此外,circ_0044411海绵作用miR-141-3p正向调节CCL16表达。
结论:Circ_0044411敲低可促进细胞活力和增殖,通过调节miR-141-3p/CCL16轴抑制炎症反应和细胞凋亡,表明circ_0044411可能是IP的潜在治疗靶标。