Anti-oxydative effects of miR-204 on LECs in age-related cataract

Background

miRNAs are a group of non-coding small RNA molecules, and they play an important role in regulating the apoptosis of LECs.The biological effects and mechanisams of miRNAs on LECs in age-related cataract still need to be further elucidated.

Objective

This study was to investigate the anti-oxidative effects of miR-204 on age-related cataract in vitro.

Methods

The specimens of anterior lens capsules from age-related cataract patients and normal donors were collected in Shandong Eye Institution and 20 subjects for each.The expression level of miR-204 was detected and compared between cataractous eyes and normol eyes by real-time fluorescence quantitative PCR (RT-qPCR). HLE-B3 cells, a human LEC line, were cultured, and the oxydative stress models of LECs were established by adding 200 μmol/L H₂O₂ in the medium.The models were divided into model control group, miR-204 agomir group, agomir negative control group, miR-204 antgomir group and antagomir negative control group according to the difference of tranfected agents, and normal cells served as the control group.Twenty-four hours after transfection, the expression levels of miR-204 mRNA in the cells of various groups were detected by RT-qPCR to varify the transfection rate.Apoptosis rate of the cells was assayed by flow cytometry.The relative expression levels of TP53INP1 mRNA and p53 mRNA as well as their proteins were detected by RT-qPCR and Western blot, respectively.

Results

The mean expression level of miR-204 was significantly lower in the anterior lens capsules of cataractous eyes than that in the normal eyes (t=14.21, P<0.05). The mean apoptosis rate of cells was 1.31±0.12, 4.90±0.28, 2.60±0.15, 4.39±0.20, 5.74±0.13 and 4.34±0.63 in the normal control group, model control group, miR-204 agomir group, agomir negative control group, miR-204 antgomir group and antagomir negative control group, respectively.The apoptosis rate was significantly increased in the model control group compared with the normal control group (t=-20.69, P<0.01) and the apoptosis rate was also increased in the miR-204 antagomir group compared with antagomir negative control group (t=3.79, P<0.05); while the apoptosis rate in the miR-204 agomir group was significantly declined in comparison with agomir negative control group (t=-12.20, P<0.01). The relative expression levels of TP53INP1 and p53 mRNA and proteins in the cells were significantly higher in the model control group than those in normal control group (mRNA: t=6.44, 11.71, both at P<0.01; protein: t=10.72, 19.40, both at P<0.01), and so were between the miR-204 antagomir group and antagomir negative control group (mRNA: t=4.07, 3.74, both at P<0.05; protein: t=7.18, 10.58, both at P<0.05). However, the expression levels of TP53INP1 and p53 mRNA and protein were significantly reduced in the miR-204 agomir group in comparison with the agomir negative control group (mRNA: t=-19.28, -10.58, both at P<0.05; protein: t=-6.50, -6.36, both at P<0.05).

Conclusions

miR-204 induces oxidative damage of age-related cataract via targeting TP53INP1, which suggests that the activation of TP53INP1-p53 signaling may be involved in the development of age-related cataract.