Zhang Chunmei, Liu Jingjing, Jin Na, Zhang Guiming, Xi Yahui, Liu Hongling
Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
Department of Ophthalmology, No.2 Hospital of Xiamen, Fujian, P.R. China.
PLoS One. 2016 Dec 2;11(12):e0167349. doi: 10.1371/journal.pone.0167349. eCollection 2016.
Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing PCO.
后囊膜混浊（PCO）是囊外白内障摘除术后导致视力下降的最常见并发症。PCO形成的主要原因是残留晶状体上皮细胞（LECs）的增殖。雷帕霉素靶蛋白（mTOR）在LECs的生长和迁移中起重要作用。在本研究中，我们使用小干扰RNA（siRNA）特异性减弱人晶状体上皮B3细胞（HLE B3）中的mTOR。我们旨在研究mTOR-siRNA对HLE B3细胞增殖、迁移和上皮-间质转化（EMT）的影响，并探索其潜在机制。使用脂质体2000将mTOR-siRNA转染到HLE B3细胞中。检测mTOR的mRNA和蛋白水平以确认mTOR-siRNA的效率。分别使用实时PCR或蛋白质印迹法检测mTOR下游效应器p70核糖体蛋白S6激酶（p70S6K）和蛋白激酶B（PKB，AKT）的mRNA和蛋白水平以及活性。使用细胞计数试剂盒（CCK）8和细胞生长曲线分析来测定细胞增殖。使用Transwell系统和划痕试验检测细胞迁移。mTOR-siRNA有效消除了mTOR的mRNA和蛋白。mTOR-siRNA转染显著抑制了细胞增殖和迁移。mTOR-siRNA以时间依赖性方式降低了p70S6K和AKT的mRNA水平。此外，mTOR-siRNA降低了p70S6K和AKT的磷酸化水平。mTOR-siRNA还消除了mTORC1和mTORC2蛋白复合物的形成，并阻断了转化生长因子（TGF）-β诱导的EMT。我们的结果表明，mTOR-siRNA可通过抑制p70S6K和AKT有效抑制HLE B3细胞的增殖、迁移和EMT。这些结果表明，mTOR-siRNA可能是一种抑制白内障手术后HLE细胞生长和EMT的有效药物，并为预防PCO提供了一种替代疗法。
