转化生长因子-β_2(TGF-β_2)诱导人视网膜色素上皮层细胞上皮-间质转分化中miRNA-29b的表达及意义

Expression changes and significance of miRNA-29b in TGF-β_2 induced epithelial mesenchymal transition of human retinal pigment epithelial cells

目的探究转化生长因子-β2(transforming growth factor-β2,TGF-β2)诱导人视网膜色素上皮(retinal pigment epithelial,RPE)层细胞上皮-间质转分化中miRNA-29b的表达变化及意义。方法使用不同浓度TGF-β2刺激RPE细胞上皮-间质转分化后,倒置相差显微镜观察细胞形态变化,Western blot、RT-PCR检测成纤维化相关分子纤维连接蛋白(fibronection,FN)、神经钙粘连蛋白(nerve calcium adhesion protein,N-Cadherin)的表达。采用RT-PCR检测不同浓度TGF-β2及不同时间TGF-β2(5μg·L^(-1))刺激RPE细胞后miRNA-29b的表达。结果 TGF-β2刺激后的RPE细胞形态呈纤维化改变。在一定浓度范围内(1μg·L^(-1)、5μg·L^(-1)、10μg·L^(-1)),随着TGF-β2浓度的增加FN、N-Cadherin及相应mRNA随之增加,1μg·L^(-1)、5μg·L^(-1)、10μg·L^(-1)组与对照组(0μg·L^(-1))相比,差异均有统计学意义(均为P<0.01)。TGF-β2在一定浓度范围内(0μg·L^(-1)、1μg·L^(-1)、5μg·L^(-1))以剂量依赖的方式诱导RPE细胞miRNA-29b表达的降低,1μg·L^(-1)、5μg·L^(-1)、10μg·L^(-1)组与对照组(0μg·L^(-1))相比,差异均有统计学意义(均为P<0.01),在TGF-β2浓度为5μg·L^(-1)时miRNA-29b表达量最低。TGF-β2在一定时间范围内(0 h、3 h、6 h、12 h)以时间依赖的方式诱导RPE细胞miRNA-29b表达的降低,3 h、6 h、12 h、24 h、48 h组与0 h相比差异均有统计学意义(均为P<0.05)。结论一定范围内TGF-β2以剂量和时间依赖的方式诱导RPE细胞miRNA-29b的表达,为进一步研究miRNA-29b与TGF-β2在RPE细胞上皮-间质转分化过程中的相互作用提供理论基础。

Objective To explore the expression changes and significance of miRNA-29b in TGF-β2 induced epithelial mesenchymal transition （EMT） of human retinal pigment epithelial （RPE） cells. Methods Different concentrations of TGF-β2 （0 μg · L^-1 ,1 μg · L^-1,5 μg · L^-1 ,10μg · L^-1） were used to stimulate EMT of RPE ceils, and then the changes of cell morphology were observed by in- verted phase contrast microscope. The expression of fibronectin （FN） and nerve calcium adhesion protein （N-Cadherin） were detected by Western blot and RT- PCR. RT-PCR was used to detect the expression of miRNA-29b in RPE cells after stimulated with TGF-β2 at different concentrations（0μg · L^-1, 1 μg · L^-1 ,5 μg · L^-1 ,10μg · L^-1） and at different time points （0 hour,3 hours,5 hours,12 hours, 24 hours,48 hours） of TGF-β2 （5 μg · L^-1 ）. Results RPE cells exhibited a fiber-like morphology after stimulated with TGF-β2. With the increase of TGF-β2 concentration （1μg · L^-1 ,5 μg · L^-1 ,10 μg · L^-1） and the expression of FN,N- Cadherin protein and the corresponding mRNA increased. Other groups （ 1 μg · L^-1 ,5 μg · L^-1, 10 μg · L^-1 ） compared with blank control group （0 μg · L^-1）, the differences were significant （ all P 〈 0. 01 ）. RPE cells induced by TGF-β2 showed a decreasing expression of miRNA-29b in a dose dependent manner （0 μg · L^-1,1μg · L^-1,5 μg · L^-1）. Other groups （1μg · L^-1,5μg · L^-1, 10 μg · L^-1 ） compared with blank control group （0 μg · L^-1 ） and the differences were significant （ all P 〈0.01 ）. When the TGF-β2 concentration was 5 μg · L^-1, the expression of miRNA-29b reached the lowest. RPE cells induced by TGF-β2 showed a decreasing expression of miRNA-29b in a time dependent manner （0 hour,3 hours,6 hours, 12 hours）. Other groups （3 hours,6 hours, 12 hours,24 hours,48 hours） compared with blank control group （0 hour） and the differences were significant （ all P 〈 0.05 ）. Conclusion TGF-β2 can induce the expression of miRNA-29b in RPE cells in a time and dose-dependent manner in a certain range,which provide a theoretical basis for the further study of the cross-talk miRNA-29b and TGF-β2 in the EMT process of RPE cells.