长链非编码RNA GM10786调控miR-142-Nrf2轴抑制糖尿病肾病单核细胞炎性因子分泌的作用及机制研究

Long non-coding RNA GM10786 inhibits the secretion of inflammatory cytokines by regulating miR-142-Nrf2 axis in diabetic nephropathy

目的探讨lncRNA GM10786靶向结合miR-142抑制糖尿病肾病(DN)单核巨噬细胞炎性因子分泌的作用及机制。方法高通量转录芯片筛选DN患者与健康对照者外周血单核细胞差异表达lncRNA。实时荧光定量PCR(qRT-PCR)检测高糖刺激下THP-1细胞中GM10786的表达水平。在THP-1细胞中转染Lv-control、Lv-GM10786后,qRT-PCR测定高糖刺激下转染后细胞中pro-IL-1β和TNF-α的表达水平。采用双荧光素酶报告基因实验验证GM10786与miR-142的靶向结合及miR-142靶向调控Nrf2。THP-1细胞中转染空质粒(Control)、miR-142、以及共转染GM10786和miR-142后,qRT-PCR和Western blotting检测Nrf2和HO-1的表达水平,ELISA和Western blotting检测高糖刺激下转染细胞培养上清及细胞中IL-1β和TNF-α的表达水平。结果与健康对照组相比,GM10786在DN患者外周血单核细胞中的表达显著下调(P<0.05)。在高糖刺激下GM10786在THP-1中的表达相较正常培养基也显著下调(P<0.05)。与HG+Lv-control组相比,HG+Lv-GM10786组中pro-IL-1β和TNF-α的表达水平均下调,差异有统计学意义(P<0.001)。双荧光素酶报告基因证实GM10786能够与miR-142特异性结合,Nrf2是miR-142的靶基因。与转染miR-142组相比,共转染GM10786和miR-142组的THP-1细胞中Nrf2和HO-1表达水平显著增加(P<0.05),高糖刺激下IL-1β和TNF-α的表达水平均显著下降(P<0.05)。结论GM10786是DN进程中重要的负调控因子,GM10786通过海绵吸附作用靶向结合miR-142和调节Nrf2表达来抑制DN中单核巨噬细胞炎性因子的分泌,该研究将有助于DN的靶向治疗。

This study was performed to investigate the inhibitory effect of lncRNA GM10786 on the secretion of inflammatory factors in monocytes of diabetic nephropathy(DN)and its mechanism.High-throughput transcription microarray was used to screen differentially expressed lncRNA in PBMCs from DN patients and healthy subjects.The expression levels of GM10786 in THP-1 cells stimulated with high glucose were measured by qRTPCR.After transfection of Lv-control or Lv-GM10786 in THP-1 cells,the expression levels of pro-IL-1βand TNF-αin transfected cells stimulated with high glucose were determined by qRT-PCR.Dual luciferase reporter assay was used to verify the targeted binding of GM10786 to miR-142 and the miR-142 targeted regulation of Nrf2.After transfection of control(empty plasmid),miR-142,or co-transfection of GM10786 and miR-142 in THP-1 cells,the expression levels of Nrf2 and HO-1 were detected by qRT-PCR and Western blotting,and the expression levels of IL-1βand TNF-αin transfected cell culture supernatant and cells stimulated with high glucose were detected by ELISA and Western blotting.Data showed that the expression of GM10786 was significantly down-regulated in PBMCs of DN patients compared with healthy controls(P<0.05);the expression of GM10786 in THP-1 stimulated by high glucose was also significantly down-regulated compared with normal medium(P<0.05).Compared with the HG+Lv-control group,the expression levels of both pro-IL-1βand TNF-αwere down-regulated in HG+Lv-GM10786(P<0.001).Dualluciferase reporter confirmed that GM10786 was able to specifically bind to miR-142,and Nrf2 is a target gene of miR-142.Compared with the group transfected with miR-142,Nrf2 and HO-1 expression levels were significantly increased in co-transfected GM10786 and miR-142 THP-1 cells(P<0.05),and the expression levels of IL-1βand TNF-αwere significantly decreased under high glucose stimulation(P<0.05).In conclusion,GM10786 is an important negative regulator in the process of DN.GM10786 inhibits the secretion and activation of monocyte and macrophage inflammatory factors in DN by targeting miR-142 and regulating Nrf2 expression through sponge adsorption.