长链非编码RNA Gm4419通过NF-κB信号通路调控高糖下系膜细胞炎症因子的表达

目的 探讨长链非编码RNA Gm4419通过核转录因子-kappa B(nuclear factor-kappa B,NF-κB)信号通路对高糖培养的肾小球系膜细胞炎症因子表达的影响.方法 在高、低糖培养的小鼠肾小球系膜细胞中,采用RT-qPCR检测Gm4419的亚细胞分布及NF-κB亚基p50和p65的表达水平;采用RT-qPCR及免疫荧光检测上、下调Gm4419后炎症相关因子TNF-α和MCP-1的表达;采用生物信息技术分析Gm4419与p50和p65关系;采用Western blot检测上、下调Gm4419后细胞核中p50和p65的表达水平,以及检测特异性p50抑制剂对过表达Gm4419影响MCP-1表达的作用.结果 Gm4419在肾小球系膜细胞高糖组表达水平显著高于低糖组(P＜0.05),且主要分布在系膜细胞的细胞质中.同时,在低糖组过表达Gm4419后MCP-1、TNF-α表达水平显著增加(P＜0.01);而在高糖组沉默Gm4419后MCP-1、TNF-α表达水平显著减少(P＜0.01).此外,Gm4419与NF-κB亚基p50和p65均存在潜在结合位点,过表达Gm4419后胞核p50和p65蛋白表达水平显著增加,NF-κB活化增强(P＜0.01);而沉默Gm4419后胞核p50和p65蛋白表达水平显著降低,NF-κB活化减弱(P＜0.01),且过表达Gm4419增加MCP-1表达的效应可被特异性p50抑制剂阻断.结论 Gm4419可能在促进高糖下系膜细胞炎症因子表达中扮演重要角色,其机制可能涉及NF-κB信号通路.

LncRNA Gm4419 regulates inflammatory cytokines expression via NF-κB signaling pathway in mesangial cells under high glucose condition

Objective To investigate the mechanism of long non-coding RNA (lncRNA) Gm4419 regulating the expression of inflammatory cytokines in glomerular mesangial cells under high glucose condition via mediating nuclear factor-kappa B (NF-κB) signaling pathway.Methods The subcellular distribution of Gm4419 and expression of NF-κB subunits p50 and p65 were detected by RT-qPCR in mouse glomerular mesangial cells (MCs) under high or low glucose condition.Moreover,RT-qPCR and immunofluorescence assay were used to measure the expression of MCP-1 and TNF-α when Gm4419 was up-or down-regulated.Additionally,bioinformatics analysis was carried out to predict the relationship between Gm4419 and NF-κB.Western blot assay was used to determine the expression of p50 and p65 in the nucleus of MCs,and the effect of specific p50 inhibitor on the expression of MCP-1 was studied in MCs with Gm4419 over-expression.Results The expression of Gm4419 was significantly higher in MCs under high glucose condition than those under low condition (P ＜ 0.05),and Gm4419 mainly located in the cytoplasm.The expression levels of MCP-1 and TNF-α were significantly increased in the low glucose group when Gm4419 was over-expressed (P ＜0.01),while the levels were decreased in high glucose group when Gm4419 was silenced (P ＜0.01).Moreover,the binding sites of Gm4419 and p50 and p65 were predicted,and over-expression of Gm4419 resulted in the increases in p50 and p65 expression levels and enhancement in NF-κB activity (P ＜ 0.01).However,silencing Gm4419 induced opposite effects (P ＜0.01).Additionally,specific p50 inhibitor could block the up-reguation of MCP-1 induced by Gm4419 over-expression.Conclusion Gm4419 may play an important role in the enhanced expression of inflammatory cytokines in mesangial cells under high glucose condition,and its mechanism may be related to the NF-κB signaling pathway.