| 刘仁海,穆伽俐,陈小莲,等.miR-126-5p通过靶向 Peli2影响糖尿病肾病肾小管上皮细胞上皮 -间质转化发生[J].安徽医药,2021,25(7):1428-1432. |
| miR-126-5p通过靶向 Peli2影响糖尿病肾病肾小管上皮细胞上皮 -间质转化发生 |
| miR-126-5p affects EMT occurrence in renal tubular epithelial cells of diabetic nephropathy by targeting Peli2 |
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| DOI：10.3969/j.issn.1009-6469.2021.07.038 |
| 中文关键词: 糖尿病肾病 泛素蛋白连接酶类 微小 RNA-126-5p pellino E3泛素蛋白连接酶家族成员 2 肾小管上皮细胞 上皮 -间质转化 |
| 英文关键词: Diabetic nephropathies Ubiquitin-protein ligases |
| 基金项目:重庆市沙坪坝区决策咨询与管理创新项目( Jcd202038) |
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| 中文摘要: |
| 目的探讨微小 RNA-126-5p(miR-126-5p)通过靶向 pellino E3泛素蛋白连接酶家族成员 2(Peli2)影响糖尿病肾病肾小管上皮细胞上皮 -间质转化(EMT)发生的分子机制。方法本研究起止时间为 2018年 12月至 2019年 12月。体外培养人肾小管上皮细胞 HK-2(美国 ATCC)，分别使用 5 mmol/L、30 mmol/L的 D-葡萄糖处理细胞 48 h，分别记作对照组、模型组。采用实时荧光定量聚合酶链反应( qRT-PCR)与蛋白免疫印迹法( Western blot)分别检测 miR-126-5p、Peli2的表达水平。利用脂质体转染法分别将 miR-126-5p寡核苷酸模拟物( miR-126-5p mimics)及阴性对照 mimic NC序列( miR-NC)、 Peli2小分子干扰 RNA(si-Peli2)及其阴性对照( si-NC)转染至 HK-2细胞，使用 30 mmol/L的 D-葡萄糖处理细胞 48 h。Western blot检测上皮型钙黏蛋白( E-Cadherin)、神经型钙黏蛋白( N-Cadherin)、波形蛋白( Vimentin)、锌指蛋白( Snail)表达量；双荧光素酶报告实验验证 miR126-5p与 Peli2的靶向关系。结果与对照组相比，模型组 miR-126-5p［(1.00±0.10)比( 0.32±0.03)］、E-Cadherin［(0.86±0.09)比(0.35±0.04)］的表达水平显著降低( P<0.05)Peli2［( 0.42±0.04)比( 1.05±0.11)］、 N-Cadherin［( 0.45±0.05)比( 0.99±0.10)］、 Vimentin［(0.32±0.03)比( 0.92±0.09)］Snail［(0±0.02)比( 0.75±0.08)］的表达水平显著升高( P<0.05)；转染 miR-126-5p mimics，与转染 miR-NC相比， E-Cadherin［4±0.03)比( 0.75±0.08)］的表达水平显著升高( P<0.05)N-Cadherin［(1.02±0.11)比( 0.53± .22，(0.3、0.05)］、 Vimentin［( 0.95±0.10)比( 0.48±0.05)］、 Snail［( 0.72±0.07)比( 0.36±0.04)］的表达水著降低( P<0.05)；转染 si-Peli2后，与转染 si-NC相比， E-Cadherin［( 0.24±0.03)比( 0.70±0.07)］的表达水平显著升高( P<0.05)， N-Cadherin［( 1.05±0.12)比平显，(0.50±0.05)］、 Vimentin［( 0.96±0.10)比( 0.38±0.04)］、 Snail［( 0.74±0.07)比( 0.40±0.04)］的表达水平显著降低( P<0.05)； miR126-5p能够特异性结合 Peli2，Peli2是 miR-126-5p的靶基因； Peli2过表达后可明显逆转 miR-126-5p过表达对高糖诱导的 HK-2细胞 EMT的影响。结论 miR-126-5p通过靶向 Peli2抑制糖尿病肾病肾小管上皮细胞 EMT发生。 |
| 英文摘要: |
| Objective To explore the molecular mechanism of miR-126-5p affecting EMT in renal tubular epithelial cells of diabetic nephropathy by targeting Peli2.Methods The start and end time of this research was from December 2018 to December 2019. Human renal tubular epithelial cells HK-2(ATCC) were cultured in vitro, and the cells were treated with D-glucose at 5 mmol / L and 30 mmol / L for 48 h, respectively. They were recorded as NC group and HG group, respectively. qRT-PCR and Western blot were used to detect the expression levels of miR-126-5p and Peli2, respectively. The liposome transfection method was used to transfect miR-126-5p mimics, miR-NC, si-Peli2, and si-NC to HK-2 cells, and the cells were treated with 30 mmol / L D-glucose for 48 h. Western blot was used to detect the expression of E-Cadherin, N-Cadherin, Vimentin, and Snail. The dual luciferase reporting experiment verified the targeting relationship between miR-126-5p and Peli2.Results Compared with the NC group, the expression levels of miR-126-5p [(1.00±0.10) vs. (0.32±0.03)] and E-Cadherin [(0.86±0.09) vs. (0.35±0.04)] in the Model group were significantly reduced (P<0.05), and the expression levels of Peli2 [(0.42±0.04) vs. (1.05±0.11)], N-Cadherin [(0.45±0.05) vs. (0.99±0.10)], Vimentin [(0.32±0.03) vs. (0.92±0.09)], and Snail [(0.22±0.02) vs. (0.75±0.08)] were significantly increased (P<0.05). After miR-126-5p mimics or si-Peli2 transfection, the expression level of E-Cadherin [(0.34±0.03) vs. (0.75±0.08)] was significantly higher than that of miR-NC or si-NC (P<0.05), the expression levels of N-Cadherin [(1.02±0.11) vs. (0.53±0.05)], Vimentin [(0.95±0.10) vs. (0.48±0.05)], Snail [(0.72±0.07) vs. (0.36±0.04)] were significantly reduced (P<0.05). After si-Peli2, the expression level of E-Cadherin [(0.24±0.03) vs. (0.70±0.07] was significantly higher than that of si-NC (P<0.05), the expression levels of N-Cadherin [(1.05±0.12) vs. (0.50±0.05)], Vimentin [(0.96±0.10) vs. (0.38±0.04)], Snail [(0.74± 0.07) vs. (0.40±0.04)] were significantly reduced (P<0.05). miR-126-5p could specifically bind to Peli2, and Peli2 was a target gene of miR-126-5p. Peli2 overexpression could significantly reverse the effect of miR-126-5p overexpression on EMT in HK-2 cells induced by high glucose.Conclusion miR-126-5p inhibits EMT in renal tubular epithelial cells of diabetic nephropathy by targeting Peli2. |
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