微小RNA-374a对高糖诱导人肾小管上皮细胞单核细胞趋化蛋白-1等炎症因子表达的影响

Effects of MicroRNA-374a on high glucose induced expression of MCP-1 and other inflammatory factors in human tubular epithelial cells

目的研究微小RNA-374a(miRNA-374a)对高糖环境下人肾小管上皮细胞(HK2)中单核细胞趋化蛋白-1(MCP-1)及相关炎症因子表达的影响,探讨miRNA-374a调控促炎因子表达介导炎症反应参与糖尿病肾病进展的可能机制。方法体外培养人近端肾小管上皮细胞系(HK2),按如下处理分组:(1)正常对照组(D-葡萄糖5.6 mmol/L);(2)甘露醇组(D-葡萄糖5.6 mmol/L+D-甘露醇24.4 mmol/L);(3)高糖组(D-葡萄糖30 mmol/L);(4)转染对照组;(5)miRNA-374a mimics转染组;(6)miRNA-374a inhibitor转染组。培养24 h收获细胞。Western印迹法检测细胞单核细胞趋化蛋白-1(MCP-1)蛋白的表达,实时荧光定量PCR(qRTPCR)法检测细胞miRNA-374a、MCP-1 mRNA的表达,ELISA法检测细胞白介素6(IL-6)、白介素18(IL-18)、肿瘤坏死因子α(TNF-α)蛋白的表达。结果与正常对照组相比,高糖组细胞miRNA-374a表达明显减少(P<0.05),MCP-1蛋白及mRNA表达明显增加,IL-6、IL-18、TNF-α蛋白的表达均增加(均P<0.05)。与高糖组相比,miRNA-374a mimics转染组细胞MCP-1蛋白及mRNA表达明显减少,IL-6、IL-18、TNF-α蛋白的表达均相应显著降低(P<0.05)。与高糖组相比,miRNA-374a inhibitor转染组细胞MCP-1蛋白及mRNA表达明显增加,IL-6、IL-18、TNF-α蛋白的表达均显著增加(P<0.05)。结论高糖环境下miRNA-374a很可能通过调控肾小管上皮细胞MCP-1等炎症因子的表达参与糖尿病肾病炎症反应的发生发展。

Objective To research the effect of microRNA-374 a（ miRNA-374 a） on the expression of monocyte chemoattractant protein 1（ MCP-1） and related inflammatory factors in human tubular epithelial cells（ HK2） under high glucose,and to explore the possible mechanisms of miRNA-374 a regulating the expression of proinflammatory factors and mediating inflammation participated the progress of diabetic nephropathy（ DN）. Methods HK2 were cultured in vitro and divided into different groups. They were（1） Normal Control Group（ D-glucose 5. 6 mmol/L）;（2） Mannitol group（ D-glucose 5. 6 mmol/L ＋ D-mannitol 24. 4 mmol/L）;（3） High glucose group（ D-glucose 30 mmol/L）;（4） Transfection control group;（5） miRNA-374 a mimics group;（6） miRNA-374 a inhibitor group. The corresponding indexes were measured at 24 th hours. Western blotting was used to detect the protein expression of MCP-1. Real Time-PCR was used to detect mRNA expression of MCP-1 and miRNA-374 a. ELISA assay was used to assess the levels of interleukin 6（ IL-6）,interleukin 18（ IL-18） and tumor necrosis factor α（ TNF-α）. Results Compared with the control group,the expression of miRNA-374 a in the high glucose group were decreased（ P〈0. 05）,the protein and mRNA expression of MCP-1 were increased,and the protein expression of IL-6,IL-18,TNF-α were all increased（ P〈0. 05）. Compared with the high glucose group,the protein and mRNA expression of MCP-1 were decreased in miRNA-374 a mimics group,the protein expression of IL-6,IL-18,TNF-α were also decreased（ P〈0. 05）. The protein and mRNA expression of MCP-1,the protein expression of IL-6,IL-18,TNF-α were increased in miRNA-374 a inhibitor group. Conclusion MiRNA-374 a may be involved in the progress of inflammatory reaction in DN by regulating the expression of MCP-1 and other inflammatory factors in human tubular epithelial cells under high glucose condition.