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

Effects of miR-21 on protein expression of extracellular matrix in human trabecular meshwork cells under oxidative stress

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H_2O_2(0μmol·L^(-1)、200μmol·L^(-1)、400μmol·L^(-1)、600μmol·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H_2O_2浓度。随后将细胞分成正常组和H_2O_2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:H_2O_2组(只用H_2O_2处理)、miR-21干预组(H_2O_2+miR-21模拟物)、miR-21对照组(H_2O_2+miR-21对照模拟物)、miR-21抑制组(H_2O_2+miR-21抑制剂)和miR-21抑制对照组(H_2O_2+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:H_2O_2组(只用H_2O_2处理)、PTEN干扰组(H_2O_2+PTEN siRNA)和干扰对照组(H_2O_2+control siRNA),检测PTEN、胞外基质蛋白和TGF-β2蛋白水平表达。结果当H_2O_2浓度≥400μmol·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,H_2O_2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H_2O_2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H_2O_2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。

Objective To investigate the effects and mechanism of miR-21 on the protein expression of extracellular matrix in human trabecular meshwork ceils （HTMCs） under oxidative stress. Methods Different concentrations of H2 O2 （0,200 μmol · L-1, 400 μmol · L- 1 , 600 μmol · L-1 ） were used to stimulate HTMCs for 1 hour, and the proper H2 O2 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： H2 O： group （ H2 O2 only）, iniR-21 treatment group （ H2 O2 ＋ miR-21 mimic ）, miR-21 control group （ H2 O2 ＋ control mimic）, miR-21 inhibitor group （ H2 O2 ＋ 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 ceils were divided into 3 groups： H2 O2 group （H2 O2 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 H2 O2 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.