摘要
目的:研究microRNA-29b(miR-29b)在血管紧张素Ⅱ诱导肾小管上皮细胞间充质转分化(epithelial-mesenchymal transition,EMT)中的角色。方法:实时定量PCR检测自发性高血压大鼠(spontaneously hypertensive rats,SHR)和Wistar-Kyoto(WKY)大鼠肾脏皮质组织miR-29b表达的差异。体外培养NRK-52E大鼠肾小管上皮细胞,给予血管紧张素Ⅱ(Ang Ⅱ)10-7mol/L(为Ang Ⅱ组),以正常培养的细胞作空白对照,实时定量PCR检测Ang Ⅱ组和正常对照组细胞miR-29b表达差异,运用WesternBolt技术和实时定量PCR技术分别检测Ang Ⅱ组和空白对照组TGF-β、α肌动蛋白(α-SMA)和I型胶原蛋白(Col Ⅰ)的表达量。进一步分别再转染miR-29b inhibitor和miR-29b mimics,建立miR-29b低表达和高表达细胞模型。低表达模型实验分三组:①空白对照组:正常培养的肾小管上皮细胞未经任何处理;②低表达组:转染miR-29b inhibitor;③阴性对照组:转染随机合成NC microRNA片段。高表达模型实验分三组:①空白对照组:肾小管上皮细胞给予Ang Ⅱ(10-7mol/L)诱导;②高表达组:肾小管上皮细胞给予Ang Ⅱ(10-7mol/L)诱导,同时转染miR-29b mimcs;③阴性对照组:肾小管上皮细胞给予Ang Ⅱ(10-7mol/L)诱导,同时转染随机合成NC microRNA片段。用流式细胞仪技术检测转染率,用实时定量PCR检测转染效果,运用Western Bolt技术和实时定量PCR技术分别检测各组中TGF-β、α-SMA和Col Ⅰ的表达量。结果:实时定量PCR检测SHR大鼠肾脏皮质组织miR-29b的表达(0.76±0.01)相比WKY大鼠(1.00±0.00)下降(P<0.05),相比空白对照组(1.00±0.00),Ang Ⅱ组miR-29b的表达(0.56±0.06)明显下降(P<0.05),TGF-β、α-SMA、Col Ⅰ的mRNA表达和蛋白表达明显升高(P<0.05)。流式细胞仪技术检测NRK-52E细胞转染率为95.14%,相比空白对照组(1.00±0.00),低表达组miR-29b表达(0.07±0.02)明显下降,高表达组miR-29b表达(38.3±8.1)明显升高(P<0.05)。在低表达模型实验中,低表达组α-SMA、TGF-β、Col Ⅰ mRNA表达和蛋白表达比空白对照组和阴性对照组均上调(P<0.05),而空白对照组和阴性对照组间差异无统计学意义(P>0.05)。在高表达模型实验中,高表达组的NRK-52E细胞α-SMA、TGF-β、Col Ⅰ的mRNA表达和蛋白表达比空白对照组和阴性对照组均下调(P<0.05),而空白对照组和阴性对照组间差异无统计学意义(P>0.05)。结论:SHR大鼠肾脏皮质组织miR-29b的表达水平比WKY大鼠下降,这可能和SHR大鼠体内的高Ang Ⅱ水平有关,miR-29b表达降低能促进肾小管上皮细胞发生EMT,Ang Ⅱ调节肾小管上皮细胞发生EMT的一个潜在的机制可能是通过miR-29b来实现的。
Objective： To investigate the effects of microRNA-29b （miR-29b） on renal epithelial-mesenchy-mal transition （EMT） induced by Angiotensin （Ang） H. Methods： The real time PCR was preformed to analyze the expression of miR-29b in renal cortex from spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats（WKY）. Cultured NRK-52E renal tubular epithelial cells were divided into two group： AngⅡ group treated with Ang Ⅱ（ 10^-7 mol]L） and blank control group with with no extra treatment. Real time PCR was preformed to analyzethe expression of miR-29b in these two groups. Then we analyzed the expression of TGF- β, α-SMA and Col I byreal time PCR and Westem Blot. Next, miR-29b was down-regulated or up-regulated in NRK-52E cells through transfection of miR-29b inhibitor and miR-29b mimics respectively. In the model of down-regulation, CulturedNRK-52E cells were divided into three groups： The first group was blank control： conventionally cultured cells with no extra treatment; The second group was down-regulation group： transfection of miR-29b inhibitor; Thethird group was negative control： transfection of its mixture with miRNA synthesized randomly. In the model of up-regulation, Cultured NRK-52E cells were divided into three groups： The first group was Blank control：treatment with Ang Ⅱ-（10^-7 mol/L）; The second group up-regulation group： treatment with Ang Ⅱ（10-7 mol/L） and transfection of miR-29b mimics; The third group was negative control： treatment with Ang Ⅱ（10.7 mol/L） andtransfection of its mixture with miRNA synthesized randomly. Flow cytometry was preformed to examine rate of transfection. Real time PCR was preformed to examine efficiency of transfection by analysis expression of miR-29b. Then we analyzed expression of TGF- β, α -SMA and Col I by real time PCR and Western Blot, Results： Real time PCR showed that the expression of miR-29b in renal cortex from SHR （0.76 ± 0.01） was lower thanWKY （1.00 ± 0.00） （P〈0.05）, and in Ang Ⅱ group （56.4 ± 0.06） was lower than blank control group （1.00 ± 0.00） （P〈0.05）. The expressions of TGF- β, α-SMA and Col I were significantly increased in Ang Ⅱ group （P〈0.05）,compared with blank control group. Flow cytometry showed that rate of transfection in NRK-52E cells was 95. 14%. The expression of miR-29b in down-regulation group （0.07 ± 0.02） significantly decreased and in up-regulation group（38.3 ± 8.1） significantly increased compared with blank control group （1.00 ± 0.00）. In the model of down-regulation, expressions of α-SMA, TGF- β and Col Ⅰ were significantly increased in down-regulation group, compared with blank and negative control group, while there was no significant difference between the latter two groups. In the model of up-regulation, expressions of α-SMA, TGF- β and Col I signifi-cantly decreased in up-regulation group, compared with blank and negative control group, while there was no significant difference between the latter two groups. Conclusion： The expression of miR-29b in renal cortex fromSHR was lower than WKY, that may be related to high lever of Ang/Ⅰ in SHR. Down-regulation of miR-29b promotes EMT in renal tubular epithelial cells, and it is very likely that Ang Ⅱ induces EMT via miR-29b.
出处
《温州医学院学报》
CAS
2013年第2期71-77,共7页
Journal of Wenzhou Medical College
基金
卫生部科研基金资助项目(wkj2008-2-031)
温州市科技发展计划项目(Y20090002)
