miRNA-184在TGF-β_2诱导人晶状体上皮细胞上皮-间质转分化中的表达

Expression of mi R-184 in TGF-β_2 induced human lens epithelial cells epithelial mesenchymal transition

目的研究miRNA-184(miR-184)在转化生长因子β_2(transforming growth factor β_2,TGF-β_2)诱导人晶状体上皮细胞上皮-间质转分化过程中的表达及意义。方法应用不同浓度(0μg·L^(-1)、1μg·L^(-1)、5μg·L^(-1)、10μg·L^(-1)、15μg·L^(-1))TGF-β2诱导人晶状体上皮细胞发生上皮-间质转分化,细胞免疫荧光和Western blot方法观察转分化相关分子波形蛋白(Vimentin)、钙黏附蛋白E(E-Cadherin)的表达;应用qRT-PCR检测miR-184在不同浓度(0μg·L^(-1)、1μg·L^(-1)、5μg·L^(-1)、10μg·L^(-1)、15μg·L^(-1))TGF-β_2、不同作用时间(0 h、6 h、12 h、24 h、48 h)TGF-β_2(10μg·L^(-1))诱导人晶状体上皮细胞转分化中的表达变化。结果随着TGF-β_2浓度的增加,细胞胞浆中Vimentin荧光强度增强;E-Cadherin蛋白表达逐渐减少,各组细胞间E-Cadherin表达量差异有统计学意义(F=87.617,P<0.01);Vimentin蛋白表达逐渐增加,各组细胞间Vimentin表达量差异有统计学意义(F=68.923,P<0.01)。随着TGF-β_2浓度的增加,细胞中miR-184相对表达量增多,在10μg·L^(-1) TGF-β_2诱导组达到峰值;随着TGF-β_2诱导时间的延长,细胞中miR-184相对表达量增加,在24 h表达达到峰值。结论 TGF-β_2诱导人晶状体上皮细胞发生上皮-间质转分化时细胞内miR-184表达增加,并与诱导浓度和时间有关,为进一步研究miR-184在人晶状体上皮细胞上皮-间质转分化中的作用及机制提供实验基础。

Objective To investigate the expression and significance of miR-184 in TGF-β_2 induced human lens epithelial cells（HLEC） epithelial mesenchymal transition（EMT）.Methods EMT in HLEC was induced by different concentrations of TGF-β_2（0 μg· L^（-1）,1 μg ·L^（-1）,5 μg· L^（-1）,10 μg · L^（-1）,15 μg · L^（-1）,and then immunofluorescence and Western blot were used to observe the expression of vimentin and E-Cadherin.Quantitative real-time PCR（qRT-PCR） was performed to analyze miR-184 expressions in TGF-β_2 induced HLEC at different concentration（0 μg · L^（-1）,1 μg · L^（-1）,5μg · L^（-1）,10 μg · L^（-1）,15μg · L^（-1）） and at different time（0 hour,6 hours,12 hours 24 hours,48 hours） of TGF-β_2（10 μg · L^（-1））.Results With the increase of TGF-β_2 concentration,the fluorescence intensity of vimentin in cytoplasm increased,E-C adherin protein gradually reduced,and the differences of E-C adherin expressions among groups were statistically significant（F = 87.617,P〈0.01）.Meanwhile,vimentin protein gradually increased,the differences of vimentin expressions among groups were statistically significant（F =68.923,P〈0.01）.With the increase of TGF-β_2 concentration,the relative expression of miR-184 in HLEC increased and reached the peak in 10 μg · L^（-1） TGF-β_2 induced group.With the extension of TGF-β_2 induction time,the relative expression of miR-184 in HLEC increased and reached the peak at 24 hours.Conclusion The expression of miR-184 in TGF-β_2 induced EMT of HLEC is increased and related to TGF-β_2 concentration and induction time,which provides the experimental basis to further study the role and mechanism of miR-184 in EMT of HLEC.