LncRNA NEAT1调控miR-22-3p对脂多糖诱导的人牙龈成纤维细胞生物学活性影响

Effect of LncRNA NEAT1 on Biological Activity of Lipopolysaccha-ride-induced Human Gingival Fibroblasts by Regulating miR-22-3p

目的探讨长链非编码RNA(long non-coding RNA,LncRNA)核富集转录本1(nuclear-enriched abundant transcript 1,Neat1)调控微小RNA miR-22-3p对脂多糖(lipopolysaccharide,LPS)诱导的人牙龈成纤维细胞(human gingival fibroblasts,HGFs)生物学活性的影响。方法取对数生长期的HGFs细胞分为对照组、LPS组、si-NC组、si-Neat1组、si-Neat1+inhibitor NC组、si-Neat1+miR-22-3p inhibitor组。RT-qPCR检测Neat1、miR-22-3p表达水平;双荧光素酶验证Neat1、miR-22-3p的靶向关系;流式细胞仪、CCK-8、ELISA法分别检测细胞凋亡、细胞活力及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)水平;蛋白质印迹检测Caspase-3、磷酸化核转录因子-κB(phosphorylated nuclear factor-κB,p-NF-κB)P65/NF-κB P65水平。结果与对照组比较,LPS组细胞活力、miR-22-3p表达显著降低,TNF-α、IL-1β、细胞凋亡率、p-NF-κB P65/NF-κB P65、Neat1、Caspase-3表达显著增加(P<0.05);与LPS组、si-NC组比较,si-Neat1组细胞活力、miR-22-3p表达显著增加,TNF-α、IL-1β、细胞凋亡率、p-NF-κB P65/NF-κB P65、Neat1、Caspase-3表达显著降低(P<0.05);下调miR-22-3p表达逆转了沉默Neat1对LPS诱导HGFs生物学活性的影响(P<0.05)。结论沉默Neat1可以抑制LPS诱导HGFs的细胞凋亡、炎症反应,可能与上调miR-22-3p表达有关。

Objective To investigate the influence of long non-coding RNA(LncRNA)nuclear-enriched abundant transcript 1(NEAT1)on the biological activity of human gingival fibroblasts(HGFs)induced by lipopolysaccharide(LPS)by regulating miR-22-3p.Methods HGFs cells in logarithmic growth phase were grouped into 6 groups:control group,LPS group,si-NC group,si-Neat1 group,si-Neat1+inhibitor NC group,and si-Neat1+miR-22-3p inhibitor group.RT-qPCR was applied to detect the expression levels of Neat1 and miR-22-3p.Dual luciferase reporter assay was applied to verify the targeting relationship of Neat1 and miR-22-3p.Flow cytometry,CCK-8,and ELISA were used to detect apoptosis,cell viability,and levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),respectively.Western blotting assay was applied to detect the expression levels of caspase-3and NF-κB P65,and the phosphorylation level of NF-κB P65(p-NF-κB P65).Results The cell viability and the expression level of miR-22-3p in the LPS group were significantly decreased,while the TNF-α,IL-1β,apoptosis rate,the levels of p-NF-κB P65/NF-κB P65,Neat1,and Caspase-3 were significantly increased(P<0.05),when compared with those in the control group.The cell viability and the expression level of miR-22-3p in the si-Neat1 group were significantly increased,while the TNF-α,IL-1β,apoptosis rate,the levels of p-NF-κB P65/NF-κB P65,Neat1and Caspase-3 were significantly decreased(P<0.05),when compared with those in the LPS group and the si-NC group.Down-regulation of miR-22-3p expression reversed the effect of Neat1 silencing on the biological activity of LPS-induced HGFs(P<0.05).Conclusion Silencing Neat1 can inhibit the apoptosis and inflammatory response of HGFs induced by LPS,the mechanism may be related to the up-regulation of miR-22-3p expression.