Effects Of MiR-204 On Oxidative Damage Of Lens Epithelial Cells In Age-related Cataracts And Its Mechanism

Objective:This study was to investigate the anti-oxidative damage effects of mi R-204 on Human Lens Epithelianl Cell(HLECs) of age-related cataract.Methods:The specimens of anterior lens capsules from age-related cataract patients and normal transparent donors were collected in Shandong Eye Institution and 20 subjects for each. mi RNA microarrarys technique was employed to dectect the differential expressioned mi RNA between two group.The expression level of genes was detected from anterior lens capsules between cataractous eyes and normol eyes by real-time fluorescence quantitative PCR(RT-q PCR) and predict the target genes of mi RNA with bioinformatics tools and dual luciferase. HLECs-B3 cells, a human LEC line, were cultured, and the oxydative stress models of LECs were established by adding 200 μM H2O2 in the medium to mimic oxidative damage of HLECs.then add transfection reagent into DF/12 cell culture fluid respectivrly,The models were divided into H2O2 group, mi R-204 agomir group, mi R-204 agomir Negative Control group, mi R-204 antgomir group and mi R-204 antagomir Negative Control group according to the diffrence of tranfected agents, and normal cells served as the control group. Twenty-four hours after transfection, the expressional levels of mi R-204 m RNA in the cells of various groups were detected by RT-q PCR to varify the transfected rate. Apoptotic rate of the cells were assayed by flow cytometry. The relative expression levels of TP53INP1 m RNA and p53 m RNA as well as their proteins were detected by RT-q PCR and Western blot, respectively.Results:The result of micro RNA analysisi microarrarys showed that the expression level of mi R-204 in anterior lens capsules from age-related cataract patients was low-expression. The result of PCR showed that the mean expression level of mi R-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 result of bioinformatics prediction and dual luciferasereport suggested TP53INP1 was one target gene of mi R-204. The mean apotosis 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, H2O2 group, mi R-204 agomir group, agomir Negative Control group, mi R-204 antgomir group and antagomir Negative Control group, respectively. Significantly elavated apoptostic rates were found in the H2O2 group compared with the normal control group(t=-20.69,P<0.01) and mi R-204 antagomir group compared with antagomir Negative Control group(t=3.79, P<0.05); while the apoptotic rate was significantly declined in the mi R-204 agomir group compared with agomir Negative Control group(t=-12.20, P<0.001). The relative expression levels of TP53INP1 and p53 m RNA and proteins in the cells were significantly higher in the model control group than in normal control group(m RNA: t=6.44, 10.72, both at P<0.01; protein: t=11.71, 19.4, both at P<0.01), and so was between the mi R-204 antagomir group and antagomir Negative Control group(m RNA: t=4.07, 7.18, all at P<0.05; protein: t=3.74, 10.58, all at P<0.05).However, the expression levels of TP53INP1 and p53 m RNA and protein were significantly reduced in the mi R-204 agomir group in comparison with the agomir Negative Control group(m RNA: t=-19.28,-19.28, all at P<0.05; protein: t=-6.50,-10.58、-6.36, all at P<0.05), while the expression of TP53INP1 and p53 m RNA in were significantly higher than, with a significant differences between them(m RNA: t=-19.28,-6.50, both at P<0.05; protein: t=-10.58,-6.36, both at P<0.05).Conclusions:mi R-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 progression of age-related cataract.