长链非编码RNA Rpph1对高糖培养的肾小球系膜细胞炎症因子表达的影响

Effect of long non-coding RNA Rpph1 on expression of inflammatory cytokines in mesangial cells under high glucose condition

目的探讨长链非编码RNA (long non-coding RNA,lncRNA) Rpph1对糖尿病肾病(diabetic nephropathy,DN)肾小球系膜细胞炎症因子的影响。方法采用荧光原位杂交(fluorescence in situ hybridization,FISH)检测Rpph1细胞定位,实时定量PCR (quantitative real-time PCR,qRT-PCR)检测Rpph1在DN小鼠肾脏组织和系膜细胞中的水平;生物信息学技术预测Rpph1的编码蛋白能力。构建Rpph1的过表达质粒,并设计合成Rpph1的siRNAs,用qRT-PCR验证其转染效率。在高糖培养的肾小球系膜细胞中转染Rpph1-siRNA;在低糖培养的肾小球系膜细胞中转染Rpph1过表达质粒,通过Western blot检测转染Rpph1的siRNA和过表达Rpph1后肿瘤坏死因子(tumor necrosis factorα,TNF-α)以及单核细胞趋化蛋白-1(macrophage cationic peptide 1,MCP-1)的表达情况。结果与正常小鼠比较,Rpph1在DN小鼠肾脏组织中显著上调(P <0. 01);同样,高糖培养的系膜细胞中Rpph1表达较低糖培养的系膜细胞显著上调(P <0. 01),其主要定位于系膜细胞的细胞质。此外,生物信息学证实Rpph1不具有编码蛋白的能力。且过表达Rpph1后炎症相关因子TNF-α和MCP-1的水平明显上调(P <0. 05);在下调Rpph1后,炎症相关因子TNF-α和MCP-1的表达也随之下降(P <0. 05)。结论在DN小鼠肾脏组织和高糖环境下的系膜细胞中lncRNA Rpph1显著高表达,且增强炎症相关因子的表达。Rpph1可能与糖尿病肾病炎症的发生相关。

Objective To investigate the effect of long non-coding RNA (lncRNA) Rpph1 on the expression of inflammatory cytokines in mouse glomerular mesangial cells (MCs) from mouse model of diabetic nephropathy (DN). Methods The subcellular distribution and expression of Rpph1 in the renal tissue of DN mice and the isolated glomerular mesangial cells (MCs) were detected by fluorescence in situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR). The coding potential of Rpph1 was evaluated by using ORF Finder and Coding Potential Calculator 2. The overexpressed plasmid and siRNA sequence of Rpph1 were constructed and designed and synthesized, and then transfected into MCs under low and high glucose conditions, respectively, the transfection effect of pcDNA3.1 (+)-Rpph1 and Rpph1 siRNAs was tested by qRT-PCR. Moreover, Western blotting was used to detect the expression of inflammatory factors TNF-α and macrophage cationic peptide 1 (MCP-1). Results The expression level of Rpph1 was significantly higher in the renal tissue of DN mice than that of healthy control (P<0.01). The molecule was mainly located in the cytoplasm of MCs, and its level was as well increased in MCs under high glucose condition than those under low glucose (P<0.01). Moreover, bioinformatics data identified that Rpph1 could not code proteins. Additionally, Rpph1 overexpression up-regulated significantly the levels of TNF-α and MCP-1 (P<0.05), while its knockdown induced the decreases in TNF-α and MCP-1 levels (P<0.05). Conclusion LncRNA-Rpph1 is highly expressed in the renal tissues of DN mice and in MCs cultured in high glucose, and its higher level enhances the expression of inflammatory cytokines. Rpph1 may be associated with the pathogenesis of inflammation in DN.