miR, microRNA; NC, unfavorable control; DAPI, 4,6-diamidino-2-phenylindole

miR, microRNA; NC, unfavorable control; DAPI, 4,6-diamidino-2-phenylindole. Expression of miR-15a-3p inhibits HLE-B3 cell migration To further investigate the effect of miR-15a-3p on HEL-B3 cell mobility identified the expression of miR-let-7b in lens epithelial cells from patients with ARC, and found that miRNA-let-7b was closely associated with the occurrence and development of ARC (14). 1 (MCL1) were also compared between transfected and wild-type HLE-B3 cells by DPA-714 western blot analysis. The results showed that transfection with the miR-15a-3p mimic significantly suppressed the proliferation of HLE-B3 cells, induced cell apoptosis and increased the proportion of early apoptotic cells. The migration of HLE-B3 cells was significantly inhibited following transfection with miR-15a-3p mimic (P<0.01), whereas cell invasion was unaffected (P>0.05). In addition, reduced protein levels of BCL2 and MCL1 were observed in the miR-15a-3p mimic-transfected HLE-B3 cells (P<0.01). In conclusion, miR-15a-3p may suppress cell proliferation and migration, and induce cell apoptosis in lens epithelial cells through inhibiting the expression of BCL2 and MCL1, which contributes to the onset of ARCs. Cell Death Detection kit, Fluorescein; Roche Diagnostics, Indianapolis, IN, USA), the cells were cultured in a humidified incubator at 37C for 60 min. Following three PBS washes, 50 l of 4,6-diamidino-2-phenylindole was added to cells, followed by incubation at 37C in the dark and another three PBS washes. The cells were examined under a fluorescence microscope and images were captured. Statistical analysis All statistical analyses were performed with SPSS for Windows version 18.0 (SPSS, Inc., Chicago, IL, USA). Data are offered as the mean standard deviation (SD). Error bars symbolize the SD of three impartial experiments. Differences between groups were compared using one-way analysis of variance with a post hoc test (LSD). All statistical analyses were two-sided, and P<0.05 was considered to indicate a statistically significant difference. Results Expression of miR-15a-3p in transfected HLE-B3 cells causes morphological changes The HLE-B3 cells were transfected with miR-15a-3p mimic (miR-15a), miR-15a-3p mock and mimic NC. No expression of miR-15a-3p was present in the NC cells, as detected by RT-qPCR analysis, whereas there was significantly elevated expression of miR-15a-3p in the miR-15a-3p mimic-transfected cells (Fig. 1A). Open in a separate window Physique 1. Expression of miR-15a-3p causes morphological changes in HLE-B3 Rabbit polyclonal to SRP06013 DPA-714 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis demonstrating the expression of miR-15a in transfected HLE-B3 cells. (B) Comparison of the morphology of miRNA-15a-expressing HLE-B3 cells with the NC and mock groups of cells. *P<0.01. miR, microRNA; NC, unfavorable control. Morphological changes occurred in the miR-15a-3p cells. The normal HLE-B3 cells were irregular polygon-shaped with interactions that created network structures. The morphology of the miR-15a-3p mimic-transfected cells began changing from 24 h post-transfection. The cells became large and round, with unclear boundaries and reduced networks (Fig. 1B). Expression of miR-15a-3p inhibits HLE-B3 cell proliferation The effect of the expression of miR-15a-3p on HLE-B3 cell proliferation was investigated using an MTT assay. From 48 h post-transfection, cell proliferation in the miR-15a group was progressively inhibited. On days 3, 4 and 5 post-transfection, the proliferation rates of the miR-15a cells were significantly lower than those of the Mock and NC cells (P<0.01) (Fig. 2A). Open in a separate window Physique 2. Expression of miR-15a-3p inhibits HLE-B3 cell proliferation. (A) MTT assay showing the proliferation of transfected HLE-B3 cells. (B) Colony formation of transfected HLE-B3 cells. (C) Cell count for the colony formation assay. *P<0.01. miR, microRNA; NC, unfavorable control; OD, optical density. The proliferation of transfected HLE-B3 cells was further confirmed using a plate colony formation assay. The results showed that, compared with DPA-714 the NC and mock cells, the colony-forming ability of the miR-15a cells was significantly attenuated (Fig. 2B and C). Expression of miR-15a-3p induces HLE-B3 cell apoptosis The apoptosis of the transfected HLE-B3 cells was investigated using a TUNEL assay. The results showed that obvious apoptotic signals were detected in the miR-15a cells compared with the mock and NC cells, suggesting that miR-15a-3p may induce HLE-B3 cell apoptosis (Fig. 3A). Following Annexin DPA-714 V-FITC/PI double staining and circulation cytometry, a significantly higher ratio of early apoptotic cells was found in the miR-15a cells than in the mock and NC cells. There was also more cell debris in the miR-15a cells than the NC cells (Fig. 3B). Open in a separate window Physique 3. Expression of miR-15a-3p induces HLE-B3 cell apoptosis. (A) Terminal deoxynucleotidyl transferase dUTP nick end labeling assay of transfected HLE-B3 cells. (B) Cell apoptotic rates detected by circulation cytometry. *P<0.01. miR, microRNA; NC, unfavorable control; DAPI, 4,6-diamidino-2-phenylindole. Expression of miR-15a-3p inhibits HLE-B3 cell migration To further investigate the effect.

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