微小RNA-142-3p靶向肿瘤坏死因子-α对脂多糖诱导的人鼻黏膜上皮细胞炎症反应的影响

Effect of microRNA-142-3p targeting tumor necrosis factor-alpha on inflammatory response of human nasal epithelial cells induced by lipopolysaccharide

目的探讨微小RNA(miR)-142-3p对脂多糖(LPS)诱导的人鼻黏膜上皮细胞(HNEPC)炎症反应的影响及其与TNF-α的靶向关系。方法选取HNEPC为研究对象,使用LPS诱导建立炎症反应模型。将miR-142-3p上调、miR-142-3p下调、空质粒转染至细胞,分为对照组、LPS组、miR-142-3p上调组、miR-142-3p下调组、空质粒组;将TNF-α下调、下调对照质粒分别与miR-142-3p上调质粒共同转染至细胞,记为miR-142-3p上调+TNF-α下调组、空质粒+TNF-α下调组、miR-142-3p上调组和空质粒组。采用四甲基偶氮唑蓝(MTT)检测细胞增殖能力;采用实时荧光定量聚合酶链反应(RT-qPCR)检测miR-142-3p和白细胞介素(IL)-6、IL-10、干扰素γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)mRNA水平;采用酶联免疫吸附试验检测细胞上清液中IL-6、IL-10、IFN-γ、TNF-α含量;通过TargetScan网站、荧光素酶报告实验分析miR-142-3p与TNF-α的靶向关系。结果LPS组细胞增殖能力高于对照组,miR-142-3p上调组细胞增殖能力高于空质粒组,miR-142-3p下调组细胞增殖能力低于空质粒组,差异有统计学意义(P<0.05)。LPS组细胞IL-6、IL-10、IFN-γ及TNF-α蛋白及其mRNA表达水平高于对照组,miR-142-3p上调组细胞相关炎性因子表达水平高于空质粒组,miR-142-3p下调组细胞相关炎性因子表达水平低于空质粒组,差异有统计学意义(P<0.05)。TNF-α下调组细胞IL-6、IL-10、IFN-γ及TNF-αmRNA及细胞上清液中蛋白表达水平低于下调对照组,差异有统计学意义(P<0.05)。miR-142-3p与TNF-α具有良好的靶向关系。结论miR-142-3p在慢性鼻窦炎伴鼻息肉患者鼻黏膜组织中高表达,过表达miR-142-3p可促进LPS诱导HNEPC炎性反应,可能与上调TNF-α基因表达有关。

Objective To observe the effect of microRNA(miR)-142-3p on lipopolysaccharide(LPS)-induced inflammatory response in human nasal mucosal epithelial cells(HNEPC)and its targeting relationship with TNF-α.Methods HNEPC were selected,and an inflammatory response model was established by LPS induction.The miR-142-3p up-regulated,miR-142-3p down-regulated and empty plasmid were transfected into cells,which were divided into control group,LPS group,miR-142-3p up-regulated group,miR-142-3p down-regulated group and empty plasmid group;TNF-αdown-regulated and down-regulated control plasmids and miR-142-3p up-regulated plasmids were co-transfected into cells,respectively,and were labeled as miR-142-3p up-regulated+TNF-αdown-regulated group,empty plasmid+TNF-αdown-regulated group,miR-142-3p up-regulated group and empty plasmid group.Methyl Thiazolyl Tetrazolium(MTT)was used to detect cell proliferation;the real-time reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the mRNA levels of miR-142-3p,interleukin(IL)-6,IL-10,interferon-γ(IFN-γ)and tumor necrosis factor-alpha(TNF-α);the contents of IL-6,IL-10,IFN-γand TNF-αin the supernatant were detected by enzyme-linked immunosorbent assay;the targeting relationship between miR-142-3p and TNF-αwas analyzed by TargetScan website and luciferase report experiment.Results The cell proliferation capacity of the LPS group was significantly higher than that of the control group,was significantly higher in the miR-142-3p up-regulated group than that of the empty plasmid group,and was significantly lower in the miR-142-3p down-regulated group than that of the empty plasmid group(P<0.05).The expression IL-6,IL-10,IFN-γand TNF-αproteins levels and their mRNA levels in the LPS group were significantly higher than those in the control group,the expression levels of cell-related inflammatory factors in miR-142-3p up-regulated group were significantly higher than those in the empty plasmid group,and the expression level of cell-related inflammatory factors in the miR-142-3p down-regulated group was significantly lower than that in the empty plasmid group(P<0.05).The mRNA expression levels of IL-6,IL-10,IFN-γand TNF-αand the protein expression levels of cell supernatant in the TNF-αdown-regulated group were significantly lower than those in the down-regulated control group(P<0.05).MiR-142-3p had a good targeting relationship with TNF-α.Conclusion The miR-142-3p is highly expressed in nasal mucosa tissues of patients with chronic rhinosinusitis with nasal polyps.Overexpression of miR-142-3p can promote LPS-induced HNEPC inflammatory response,which may be related to up-regulation of gene expression.