Inhibitory effects of microRNA-133b on ultraviolet-induced apoptosis of lens epithelial cells and its mechanism

Authors: Li Xiaotong,  Qin Yu,  Zhao Jiangyue,  Min Rui,  Zhang Jinsong

DOI: 10.3760/cma.j.issn.2095-0160.2017.11.005
Published 2017-11-10
Cite as Chin J Exp Ophthalmol, 2017,35(11): 977-983.

Abstract

Background

Ultraviolet B (UVB) is one of the main causes of cataract formation, and its mechanism is associated with the apoptosis of lens epithelial cells (LECs). MiroRNA-133b (miR-133b) can regulate oxidative stress-induced LECs apoptosis.However, whether miR-133b is associated with UVB-induced cataract is not elucidated.

Objective

This study was to observe the inhibitory effects of miR-133b on UVB-induced cataract and its regulating mechanism.

Methods

Twenty 8-week-old C57BL/6 mice were randomized into cataract model group and normal control group.The mouse eyes in the cataract model group exposed to 302 nm UVB for 5 minutes once per day for consecutive 1 week, with the irradiation intensity of 300 W/cm2, and the mice in the normal control group did not receive any intervention.Five mice in each group were sacrificed and 10 eyeball sections were prepared.Human LECs (SRA01/04) were exposed to UVB for 25 minutes and served as UVB-induced group, the cells in the normal control group did not receive any intervention.The UVB-induced cells were inoculated to 96-well plate and divided into 4 groups, and 50 nmol/L miR-133b mimic, miR-133b mimic control agent, miR-133b inhibitor and miR-133b inhibitor control agent were transfected into the cells with lipofectamine2000.The expression of miR-133b mRNA and a target gene BCL2L2, which was identified by online miRNA database (www.mirab.org) in the cells were detected by real-time quantitative PCR to evaluate the transfected efficacy, and the apoptosis of the cells in mouse lens tissue and different transfected groups were assayed by TUNEL.The use and care of the mice followed ARVO Statement.

Results

The arrangement of LECs was regular and no apoptotic cell was seen in the normal control group, and the apoptotic cells showed the red fluorescence in the cataract model group.The apoptotic rate of human LECs was (43.90±9.30)% in the UVB-induced group, and that in the normal control group was (1.08±0.49)%, showing a significant difference between the two groups (t=-7.963, P=0.015). The relative expression levels of miR-133b mRNA in the model mouse lens and UVB-induced human LECs were evidently lower and relative expression levels of BCL2L2 mRNA were higher than those in normal mice and normal LECs (miR-133b mRNA: t=-2.958, P=0.042; t=-6.195, P=0.003; BCL2L2 mRNA: t=3.761, P=0.020; t=12.437, P=0.000). The relative expression level of miR-133b mRNA was significantly increased and the relative expression level of BCL2L2 mRNA was reduced in the miR-133b mimic group in comparation with the miR-133b mimic control group (t=10.883, -5 927.617; both at P<0.01); compared with the miR-133b inhibitor control group, the relative expression level of miR-133b mRNA was significantly decreased and that of BCL2L2 mRNA was evidently increased in the miR-133b inhibitor group (t=-1 606.622, 17.556; both at P<0.01). The apoptotic rate of human LECs was (43.62±9.19)% and (17.55±4.24)% in the miR-133b mimic control group and miR-133b mimic group, with a significant difference between them (t=-4.462, P=0.011), and the apoptotic rate in the miR-133b inhibitor group was (78.23±12.42)%, which was significantly higher than (48.01±9.68)% in the miR-133b inhibitor control group (t=3.324, P=0.029).

Conclusions

miR-133b can prevent UVB-induced cataract probably by negatively targeting the BCL2L2 expression to regulate the apoptosis of LECs.

Key words:

MicroRNAs; Ultraviolet rays/adverse effects; Radiation injuries/pathology; Cataract/prevention & control; Epithelial cells, lens; Mice, inbred C57BL; MicroRNA-133b

Contributor Information

Li Xiaotong
Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Eye Hospital of China Medical University, Key Lens Research Laboratory of Liaoning Province, Shenyang 110005, China
Qin Yu
Zhao Jiangyue
Min Rui
Zhang Jinsong
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Updated: September 4, 2019 — 2:10 pm