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Weirong Chen; MiR-34a induce human lens epithelial cell apoptosis through E2F3 pathway. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2640.
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© ARVO (1962-2015); The Authors (2016-present)
Previous results showed miR-34a significantly expressed higher in cataract lens epithelial cells than transparent lens. To testify miR-34a induce lens epithelial cell apoptosis through E2F3 and initiate cataractogenesis.
Using stem-loop RT-PCR, testifying miR-34a expressed higher in cataract than transparent lens. Target prediction for miR-34a was performed using 4 microRNA target prediction tools, miRanda, miRWalk, PicTar5 and Targetscan. Predicted target E2F3 was experimentally validated by luciferase reporter assays in 293T cells. Human lens epithelial cells (SRA-01/04) were transfected with miRNA mimic 34a, mimic control. Apoptosis and gene expression assessed by flow cytometry and western-blot respectively.
miR-34a expressed more than 100 times in cataract lens epithelial cells compared to transparent lens epithelial cells. In 293T cells, miR-34a reduced the rellina activity from the reporter construct containing the E2F3 site(1.87±0.13), whereas no effect was observed with a construct containing a mutated E2F3 seed site(3.46±0.09). The SRA01/04 apoptosis rate, assessing by flow cytometry, showed significant higher in transfected with miR-34a(27.63%±5%) and siE2F3(28.5%±3.3%) compared to mimic control(9%±0.5%) or mock(8.37%±0.06%). Western blot analysis of SRA01/04 cells transfected with miR-34a showed a signiﬁcant reduction in E2F3 protein compared to mock and mimic control, demonstrating that miR-34a directly targets E2F3 mRNA, which was in agreement with SRA01/04 transfected with siE2F3.
miR-34a is highly expressed in cataract lens epithelial cells. And miR-34a induce human lens epithelial cell apoptosis through E2F3 pathway, which may initiate cataratogenesis.
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