01 November 2016 : Laboratory Research
Down-Regulation of MicroRNA-133b Suppresses Apoptosis of Lens Epithelial Cell by Up-Regulating BCL2L2 in Age-Related CataractsFeng ZhangABCDEFG, Weizhe MengBDE, Bin TongBCD
Med Sci Monit 2016; 22:4139-4145
BACKGROUND: MicroRNA-133b (miR-133b) has been reported to be involved in many diseases, including ovarian cancer and osteosarcoma. Accumulating evidence suggests that miR-133b plays important roles in human disease. In this study, we aimed to investigate the molecular mechanism, including the potential regulator and signaling pathways, of BCL2L2.
MATERIAL AND METHODS: We first searched the online miRNA database (www.mirdb.org) using the “seed sequence” located within the 3’-UTR of the target gene, and then performed luciferase assay to test the regulatory relationship between miR-133b and BCL2L2. Western blot and real-time PCR were used to determine the expression of BCL2L2 in human samples or cells treated with miRNA mimics or inhibitors. Flow cytometry was conducted to evaluate the apoptosis status of the cells.
RESULTS: We validated BCL2L2 to be the direct gene using a luciferase reporter assay. We also conducted real-time PCR and Western blot analyses to study the mRNA and protein expression level of BCL2L2 among different groups (control: n=29, cataract: n=33) or cells treated with scramble control, miR-133b mimics, BCL2L2 siRNA, and miR-133b inhibitors, and identified the negative regulatory relationship between miR-133b and BCL2L2. We also conducted experiments to investigate the influence of miR-133b and BCL2L2 on the viability and apoptosis of cells. The results showed that miR-133b positively interfered with the viability of cells, while BCL2L2 negatively interfered with the viability of cells, and that miR-133b inhibited apoptosis while BCL2L2 accelerated apoptosis.
CONCLUSIONS: BCL2L2 was the virtual target of miR-133b, and we found a negative regulatory relationship between miR-133b and BCL2L2. MiR-133b and BCL2L2 interfered with the viability and apoptosis of cells.
Keywords: Apoptosis - genetics, 3' Untranslated Regions, Apoptosis Regulatory Proteins - metabolism, Cataract - metabolism, Epithelial Cells - metabolism, Lens, Crystalline - metabolism, RNA, Messenger - metabolism, RNA, Small Interfering - genetics, Signal Transduction
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