lncRNA TUG1吸附microRNA-144对狼疮肾炎小鼠肾小球系膜细胞炎症因子分泌与凋亡的影响及其机制研究

Effect of lncRNA TUG1 adsorbed microRNA-144 on inflammatory factor secretion and apoptosis of glomerular mesangial cells in lupus nephritis mice and its mechanism

摘要:

目的 探讨长链非编码RNA牛磺酸上调基因1（lncRNA TUG1）吸附microRNA-144（miR-144）对狼疮肾炎小鼠肾小球系膜细胞炎症因子分泌与凋亡的影响机制。方法 B6.MRL-Fas^lprNju系统性红斑狼疮模型小鼠20只和C57BL/6健康小鼠5只在适应性条件下喂养5 d。每天收集B6.MRL-Fas^lprNju系统性红斑狼疮模型小鼠24 h尿量，尿蛋白浓度> 1 mg/L表明狼疮肾炎发病，为狼疮肾炎组。C57BL/6小鼠为正常组。分离纯化两组小鼠肾小球系膜细胞。对lncRNA TUG1实施亚细胞定位，采用实时荧光定量聚合酶链反应（qRT-PCR）检测狼疮肾炎组和正常组小鼠肾组织中lncRNA TUG1和miR-144 mRNA相对表达量。狼疮肾炎组小鼠肾小球系膜细胞转染并分为NC组（肾小球系膜细胞转染阴性对照序列）、TUG1过表达组（肾小球系膜细胞转染TUG1）、sh-TUG1组（肾小球系膜细胞转染sh-TUG1）、miR-144 mimic组（肾小球系膜细胞转染miR-144 mimic）、TUG1过表达+miR-144 mimic组（肾小球系膜细胞转染TUG1和miR-144 mimic）。双荧光素酶报告实验验证lncRNA TUG1和miR-144的靶向关系，采用酶联免疫吸附试验（ELISA）检测各组细胞中炎症因子TNF-α、IL-1β、IL-6的水平。Western blotting法检测纤维化标记因子Ⅳ型胶原（Col Ⅳ）、纤连蛋白（FN）的蛋白相对表达量。MTT法检测各组细胞增殖活力，Transwell实验检测各组细胞侵袭数，流式细胞术检测各组细胞凋亡率。结果 与正常组小鼠比较，狼疮肾炎组小鼠肾组织中的miR-144 mRNA相对表达量升高，lncRNA TUG1 mRNA相对表达量降低（P <0.05）。lncRNA TUG1与miR-144存在靶向结合关系。NC组、TUG1过表达组、sh-TUG1组、miR-144 mimic组、TUG1过表达+miR-144 mimic组细胞TNF-α、IL-1β、IL-6水平比较，差异有统计学意义（P <0.05）；与NC组比较，TUG1过表达组TNF-α、IL-1β和IL-6水平降低，miR-144 mimic组TNF-α、IL-1β、IL-6水平升高（P <0.05）。各组的Col Ⅳ、FN蛋白相对表达量比较，差异有统计学意义（P <0.05）；与NC组比较，TUG1过表达组Col Ⅳ和FN蛋白相对表达量降低，miR-144 mimic组Col Ⅳ、FN蛋白相对表达量升高（P <0.05）。各组不同时间点的肾小球系膜细胞增殖活力比较，采用重复测量设计的方差分析，结果 ①不同时间点的肾小球系膜细胞活力有差异（P <0.05）；②各组肾小球系膜细胞活力有差异（P <0.05）；③各组的细胞活力变化趋势有差异（P <0.05）；与NC组48 h和72 h比较，TUG1过表达组48 h和72 h细胞增殖活力降低（P <0.05），sh-TUG1组48 h和72 h细胞增殖活力增强（P <0.05），miR-144 mimic组48 h和72 h细胞增殖活力增强（P <0.05）。各组肾小球系膜细胞侵袭数和细胞凋亡率比较，差异有统计学意义（P <0.05）；与NC组比较，TUG1过表达组的细胞侵袭数减少（P <0.05），细胞凋亡率升高（P <0.05）；sh-TUG1组细胞侵袭数增多（P <0.05），细胞凋亡率降低（P <0.05）；miR-144 mimic组细胞侵袭数增多（P <0.05）、细胞凋亡率降低（P <0.05）。结论 lncRNA TUG1吸附miR-144进而抑制狼疮肾炎小鼠肾小球系膜细胞炎症因子分泌，减少细胞增殖并促进凋亡。

Abstract:

Objective To explore the underlying mechanism of lncRNA TUG1 in the inflammatory factors secretion and apoptosis of mesangial cells in lupus nephritis (LN) mice through adsorbing microRNA-144 (miR-144).Methods LN model mice and normal mice were purchased. The mesangial cells of model mice were isolated and purified for cell culture and treatment. Subcellular localization of lncRNA TUG1 was performed, and the fluorescence quantitative PCR was adopted to detect the expression of lncRNA TUG1 and miR-144 in LN mice and normal mice. The cells were transfected and then divided into different groups. Luciferase reporter assay was used to verify the targeting relationship between lncRNA TUG1 and miR-144. The expression of TNF-α, IL-1β, and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). Western blotting was performed to measure the protein expression of fibrosis markers. The cells proliferation, invasion, and apoptosis were respectively detected by MTT assay, Transwell assay, and flow cytometry assay.Results Compared to normal mice, the expression of miR-144 in renal tissue of LN mice was increased (P < 0.05), while the expression of lncRNA TUG1 was decreased (P <0.05). There is a targeting relationship between lncRNA TUG1 and miR-144. Cell experiment showed that compared with NC group, overexpression of TUG1 decreased the secretion of TNF-α (P < 0.05), IL-1β (P < 0.05), and IL-6 (P < 0.05), significantly down-regulated the protein expression of fibrosis marker Col.Ⅳ (P < 0.05) and FN (P < 0.05), reduced the activity of mesangial cells at 48 h (P < 0.05) and 72 h (P < 0.05), declined invasion number (P < 0.05), and enhanced apoptosis (P < 0.05). Compared with NC group, the secretion of inflammatory factors [TNF-α (P < 0.05), IL-1β (P < 0.05), and IL-6 (P < 0.05)] and the protein expression of fibrosis marker [Col.Ⅳ (P < 0.05) and FN (P < 0.05)] in miR-144 mimic group were rised, the activity of mesangial cells was enhanced at 48 h (P < 0.05) and 72 h (P < 0.05), the invasion was increased (P < 0.05), and the apoptosis was reduced (P < 0.05). The effect of lncRNA TUG1on mesangial cells of LN mice could be saved by miR-144.Conclusion Based on the results observed during this study, it quickly became apparent that lncRNA TUG1 can sponge miR-144, and inhibit the secretion of inflammatory factors in mesangial cells of LN mice, reduce cell proliferation, and promote apoptosis.