[1]党晓洁,任梅,朱江,等.氧化应激条件下miR-21对人眼小梁网细胞胞外基质蛋白表达的影响[J].眼科新进展,2017,37(1):030-34.[doi:10.13389/j.cnki.rao.2017.0008]
　DANG Xiao-Jie,REN Mei,ZHU Jiang,et al.Effects of miR-21 on protein expression of extracellular matrix in human trabecular meshwork cells under oxidative stress[J].Recent Advances in Ophthalmology,2017,37(1):030-34.[doi:10.13389/j.cnki.rao.2017.0008]
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氧化应激条件下miR-21对人眼小梁网细胞胞外基质蛋白表达的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
37卷
期数:
2017年1期
页码:
030-34
栏目:
实验研究
出版日期:
2017-01-05

文章信息/Info

Title:
Effects of miR-21 on protein expression of extracellular matrix in human trabecular meshwork cells under oxidative stress
作者:
党晓洁; 任梅; 朱江; 许治国; 杨新光
710004　陕西省西安市，陕西省眼科诊疗中心，西安市第四医院眼科，西安交通大学医学院附属广仁医院眼科
Author(s):
DANG Xiao-Jie; REN Mei; ZHU Jiang; XU Zhi-Guo; YANG Xin-Guang
Guangren Hospital Affiliated to School of Medicine of Xi’an Jiaotong University,Xi’an No.4 Hospital,Shaanxi Ophthalmic Medical Center,Xi’an 710004,Shaanxi Province,China
关键词:
miR-21; 转化生长因子; 小梁网细胞; 胞外基质
Keywords:
miR-21; PTEN; transforming growth factor; trabecular meshwork cells; extracellular matrix
分类号:
R775
DOI:
10.13389/j.cnki.rao.2017.0008
文献标志码:
A
摘要:
目的　探讨氧化应激条件下miR-21对人眼小梁网细胞(human trabecular meshwork cells,HTMCs)胞外基质蛋白表达的影响。方法　用不同浓度H2O2 (0 μmol·L-1、200 μmol·L-1、400 μmol·L-1、600 μmol·L-1)刺激HTMCs 1 h，MTT法检测其对HTMCs活力的影响，从而确定后续实验所需H2O2浓度。随后将细胞分成正常组和H2O2组，Real-time PCR检测miR-21的表达，Western blot检测胞外基质蛋白(纤维连接蛋白和胶原蛋白I)的表达。然后将细胞分成5组：H2O2组(只用H2O2处理)、miR-21干预组(H2O2+miR-21模拟物)、miR-21对照组(H2O2+miR-21对照模拟物)、miR-21抑制组(H2O2+miR-21抑制剂)和miR-21抑制对照组(H2O2+miR-21抑制剂对照物)，Real-time PCR检测miR-21、转化生长因子(transforming growth factor,TGF)-β2和PTEN mRNA表达，Western blot检测胞外基质蛋白、TGF-β2和PTEN蛋白表达。最后将细胞分成3组：H2O2组(只用H2O2处理)、PTEN干扰组(H2O2+PTEN siRNA)和干扰对照组(H2O2+control siRNA)，检测PTEN、胞外基质蛋白和TGF-β2蛋白水平表达。结果　当H2O2浓度≥400 μmol·L-1时，可显著抑制HTMCs的活性，后续实验选择此浓度。与正常组相比，H2O2组中miR-21、纤维连接蛋白和胶原蛋白I的表达均增加，差异均有统计学意义(均为P<0.05)。检测氧化应激条件下miR-21对胞外基质蛋白、TGF-β2和PTEN表达的影响，发现与H2O2组相比，miR-21对照组和miR-21抑制对照组中miR-21、纤维连接蛋白与胶原蛋白I蛋白水平表达以及TGF-β2和PTEN mRNA及蛋白水平表达差异均无统计学意义(均为P>0.05)；与miR-21对照组相比，miR-21干预组miR-21、纤维连接蛋白和胶原蛋白I蛋白水平及TGF-β2 mRNA和蛋白水平表达均增加，PTEN蛋白水平表达降低，差异均有统计学意义(均为P<0.05)，而PTEN mRNA表达差异均无统计学意义(均为P>0.05)；与miR-21抑制对照组相比，miR-21抑制组miR-21、纤维连接蛋白和胶原蛋白I蛋白水平及TGF-β2 mRNA和蛋白水平表达均降低，PTEN蛋白水平表达增加，差异均有统计学意义(均为P<0.05)，而PTEN mRNA水平差异无统计学意义(P>0.05)。采用Western blot检测氧化应激条件下PTEN对TGF-β2和胞外基质蛋白表达的影响，发现与H2O2组相比，干扰对照组PTEN、TGF-β2、纤维连接蛋白和胶原蛋白I的表达差异均无统计学意义(均为P>0.05)；与干扰对照组相比，PTEN干扰组PTEN的表达下调，TGF-β2、纤维连接蛋白和胶原蛋白I的表达均升高，差异均有统计学意义(均为P<0.05)。结论　氧化应激条件下miR-21可增加HTMCs胞外基质产物，这可能与其靶向沉默PTEN基因，调节TGF-β2的表达相关。
Abstract:
Objective　To investigate the effects and mechanism of miR-21 on the protein expression of extracellular matrix in human trabecular meshwork cells (HTMCs) under oxidative stress.Methods　Different concentrations of H2O2 (0,200 μmol·L-1,400 μmol·L-1,600 μmol·L-1) were used to stimulate HTMCs for 1 hour,and the proper H2O2 concentration in the following study was analyzed by evaluating cell viability via MTT assay.The cells were firstly divided into 2 groups:normal control group and H2O2 group.miR-21 expression was determined by Real-time PCR,and the protein expression of extracellular matrix protein (Fibronectin and Collagen I) was detected by Western blot.Next,the cells were randomly divided into 5 groups:H2O2 group (H2O2 only),miR-21 treatment group (H2O2+miR-21 mimic),miR-21 control group (H2O2+control mimic),miR-21 inhibitor group (H2O2+miR-21 inhibitor),and miR-21 inhibitor control group (H2O2+miR-21 inhibitor negative control).The mRNA expression of miR-21,transforming growth factor (TGF)-β2,and PTEN was analyzed by real-time PCR,and the protein levels of fibronectin,collagen I,TGF-β2 and PTEN were examined by Western blot.Additionally,the cells were divided into 3 groups:H2O2 group (H2O2 only),PTEN siRNA group (H2O2+PTEN siRNA),and control siRNA group (H2O2+control siRNA).The expression of PTEN,fibronectin,collagen I,and TGF-β2 was assayed by Western blot.Results　H2O2 concentrations ≥ 400 μmol·L-1,which was used in the following study,significantly inhibited the viability of HTMCs.The expression of miR-21 and extracellular matrix protein was remarkably increased in H2O2 group than in normal control group (all P<0.05).Compared with miR-21 control group,the expression of miR-21,TGF-β2,and extracellular matrix protein was significantly up-regulated (all P<0.05),PTEN protein expression was decreased (P<0.05) while the mRNA level was not changed in miR-21 treatment group.The expression of miR-21,TGF-β2,and extracellular matrix protein was markedly down-regulated (all P<0.05),PTEN protein level was enhanced (P<0.05) while the mRNA level was not altered in miR-21 inhibitor group than in miR-21 inhibitor control group.PTEN expression was decreased,while the expression of TGF-β2 and extracellular matrix protein was notably increased in PTEN siRNA group than in control siRNA group (all P<0.05).Conclusion　MiR-21 induces extracellular matrix production in HTMCs under oxidative stress,which may be related with silencing PTEN and regulating TGF-β2 expression.

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更新日期/Last Update: 2017-02-15