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小干扰RNA-Pax6对人晶状体上皮细胞生物学行为和上皮-间质转化的抑制作用
郑宇星
杨筱曦
易果果
吴淑端
冯志贞
夏朝霞
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20210423-00272
Inhibitory effect of siRNA-Pax6 on biological behavior and epithelial-mesenchymal transition of human lens epithelial cells
Zheng Yuxing
Yang Xiaoxi
Yi Guoguo
Wu Shuduan
Feng Zhizhen
Xia Zhaoxia
Authors Info & Affiliations
Zheng Yuxing
Department of Ophthalmology, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China
Yang Xiaoxi
Department of Ophthalmology, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China
Yi Guoguo
Department of Ophthalmology, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China
Wu Shuduan
Department of Ophthalmology, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China
Feng Zhizhen
Department of Ophthalmology, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China
Xia Zhaoxia
Department of Ophthalmology, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China
·
DOI： 10.3760/cma.j.cn115989-20210423-00272
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摘要

目的探讨沉默人类同源配对盒基因6(Pax6)对人晶状体上皮细胞(LECs)的生物学行为和上皮-间质转化(EMT)的作用。

方法将SRA01/04人LECs分为小干扰RNA-Pax6(siRNA-Pax6)组和siRNA阴性对照(siRNA-NC)组，siRNA-Pax6组细胞转染siRNA-Pax6，siRNA-NC组细胞转染无序siRNA。转染后24、48、72 h采用细胞计数试剂盒8法检测细胞存活率；转染后48 h，采用流式细胞术检测细胞凋亡比例和不同细胞周期细胞比例；转染后24 h，采用细胞划痕实验检测细胞迁移率；转染后48 h，采用实时荧光定量PCR检测细胞内Pax6、α-晶状体蛋白A(CRYAA)、α-晶状体蛋白B(CRYAB)、转录因子Sox2及EMT相关分子平滑肌肌动蛋白(α-SMA)、钙黏附蛋白E(E-cadherin)mRNA相对表达量，采用Western blot法检测各组细胞中Pax6蛋白相对表达量。

结果2个组转染后不同时间点细胞存活率总体比较差异均有统计学意义( F 分组＝4.776， P<0.05； F 时间＝13.535， P<0.05)，其中转染后48 h和72 h，siRNA-Pax6组细胞存活率均明显低于siRNA-NC组，差异均有统计学意义(均 P<0.05)。与siRNA-NC组比较，siRNA-Pax6组G 0/G 1期细胞比例明显增加，S期细胞比例明显减少，差异均有统计学意义( t＝9.971、-5.063，均 P<0.05)。siRNA-Pax6组细胞迁移率为(19.73±6.07)%，低于siRNA-NC组的(70.56±2.97)%，差异有统计学意义( t＝-7.245， P<0.05)。siRNA-Pax6组细胞中Sox2和α-SMA mRNA相对表达量低于siRNA-NC组，E-cadherin mRNA相对表达量高于siRNA-NC组，差异均有统计学意义( t＝-23.254、-5.294、6.062，均 P<0.01)。siRNA-Pax6组细胞中CRYAA和CRYAB mRNA相对表达量明显高于siRNA-NC组，差异均有统计学意义( t＝5.521、8.270，均 P<0.01)。siRNA-Pax6组细胞中Pax6 mRNA相对表达量为0.27±0.01，低于siRNA-NC组的1.00±0.05，差异有统计学意义( t＝-14.456， P<0.001)；siRNA-Pax6组细胞中Pax6蛋白相对表达量为0.24±0.05，低于siRNA-NC组的1.14±0.10，差异有统计学意义( t＝-4.458， P<0.001)。

结论沉默Pax6可抑制人LECs的增生及EMT过程，维持人LECs内正常晶状体蛋白的表达。

小干扰RNA;人类同源配对盒基因6;后发性白内障;人晶状体上皮细胞;上皮-间质转化
ABSTRACT

ObjectiveTo explore the effect of knockdown of the homeobox gene paired-box 6 ( Pax6) on the biological behavior and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs).

MethodsThe SRA01/04 human LECs were divided into small interfering RNA-Pax6 (siRNA-Pax6) group transfected with siRNA-Pax6 and siRNA negative control (siRNA-NC) group transfected with disordered siRNA.Cell survival rate was detected by cell counting kit-8 method at 24, 48 and 72 hours after transfection.Cell cycle distribution and apoptosis were analyzed by flow cytometry at 48 hours after transfection.Migratory capability of cells was examined by cell scratch test at 24 hours after transfection.The mRNA relative expression levels of Pax6, α-crystallin A (CRYAA), α-crystallin B (CRYAB), Sox2, α-smooth muscle actin (α-SMA) and E-cadherin were detected by quantitative real-time PCR at 48 hours after transfection.The relative expression of Pax6 protein was detected by Western blot at 48 hours after transfection.

ResultsThere was a significant difference in cell survival rates at different time points between the two groups ( F group=4.776, P<0.05; F time=13.535, P<0.05). The cell survival rate of siRNA-Pax6 group was obviously lower than that of siRNA-NC group at 48 and 72 hours after transfection, and the differences were statistically significant (both at P<0.05). Compared with siRNA-NC group, the proportion of cells in G 0/G 1 phase was significantly increased and the proportion of cells in S phase was significantly reduced in siRNA-Pax6 group ( t=9.971, -5.063; both at P<0.05). The cell migration rate of siRNA-Pax6 group was (19.73±6.07)%, which was lower than (70.56±2.97)% of siRNA-NC group, showing a statistically significant difference ( t=-7.245, P<0.05). The relative expressions of Sox2 mRNA and α-SMA mRNA were lower, and the relative expression of E-cadherin mRNA was higher in siRNA-Pax6 group than siRNA-NC group, with statistically significant differences between them ( t=-23.254, -5.294, 6.062; all at P<0.01). The relative expression of CRYAA mRNA and CRYAB mRNA was significantly higher in siRNA-Pax6 group than siRNA-NC group, and the differences were statistically significant ( t=5.521, 8.270; both at P<0.01). The relative expressions of Pax6 mRNA and protein in siRNA-Pax6 group were 0.27±0.01 and 0.24±0.05, respectively, which were both lower than 1.00±0.05 and 1.14±0.10 in siRNA-NC group, showing statistically significant differences ( t=-14.456, -4.458; both at P<0.001).

ConclusionsSilence of Pax6 can suppress the proliferation and EMT of human LECs and enhance the expression of crystallin.

RNA, small interfering;Homeobox gene paired-box 6;Posterior capsule opacification;Lens epithelial cells, human;Epithelial-mesenchymal transition
Xia Zhaoxia, Email: nc.defudabe.usys.liamxoahzaix
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引用本文

郑宇星,杨筱曦,易果果,等. 小干扰RNA-Pax6对人晶状体上皮细胞生物学行为和上皮-间质转化的抑制作用[J]. 中华实验眼科杂志,2022,40(06)：499-506.

DOI:10.3760/cma.j.cn115989-20210423-00272

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白内障是世界范围内的主要致盲眼病之一，尽管晶状体超声乳化术已广泛用于临床且术后效果较好，但术后后发性白内障(posterior capsule opacification，PCO)发生率高，可导致视力再次下降 [ 1 , 2 ]。白内障术后残留晶状体上皮细胞(lens epithelial cells，LECs)的增生、迁移和上皮-间质转化(epithelial-mesenchymal transition，EMT)是PCO的主要病理机制 [ 3 ]。人类同源配对盒基因6(homeobox gene paired-box 6，Pax6)对眼球的正常发育至关重要 [ 4 ]，目前研究认为 Pax6是晶状体形成的1个"主宰基因"，脊椎动物晶状体的形成需要大量的细胞外信号通路和基因调控网络，这些信号通路和基因调控网络由Pax6整合和执行 [ 5 ]。LECs来源于胚胎期晶状体泡前壁细胞，胚胎发育过程中部分LECs增生分化产生次级晶状体纤维细胞，其余LECs覆盖晶状体前表面。本研究团队早期实验亦证实，Pax6在早期发育的晶状体细胞中均有表达，晶状体形成后，Pax6在LECs中的表达保持不变，在晶状体纤维细胞中随纤维细胞的分化成熟表达水平逐渐下降，提示Pax6参与LECs的增生和分化 [ 6 , 7 , 8 , 9 ]。LECs具有持续自我更新和对外部损伤及氧化损伤的保护能力，是晶状体再生的功能基础。研究表明，Pax6蛋白在LECs和新生晶状体纤维中存在高表达，与正常晶状体发育过程类似，提示Pax6对LECs增生和分化的影响在晶状体损伤后依然存在 [ 10 , 11 ]。但Pax6对人LECs的相关生物学行为，尤其是对EMT的调控作用尚未完全阐明。了解Pax6在人LECs增生、迁移及EMT中的作用对PCO的防治具有重要意义。本研究拟探讨沉默Pax6对人LECs生物学行为的影响及其可能机制，以期为PCO的防治研究提供新的靶点。
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备注信息
A
夏朝霞，Email： nc.defudabe.usys.liamxoahzaix
B

郑宇星：设计实验、实施研究、采集数据、分析/解释数据、起草及修改文章；杨筱曦：设计实验、分析/解释数据；易果果：分析/解释数据；吴淑端、冯志贞：实施研究；夏朝霞：参与选题、设计实验、对文章智力性内容的修改及最终定稿

C
所有作者均声明不存在利益冲突
D
广东省自然科学基金项目 (2017A030313770)
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