Figure 6.

Differentiation of PC12 and NT2N cells were associated with modulated levels of miR-16, let-7a and miR-34a expression. PC12 cells were differentiated upon NGF treatment at 1 day with either 5 or 50 ng/ml. NGF-untreated cells were also prepared. Cells were collected at 1, 2 4 and 7 days. NT2N cells were induced to differentiate with 10 μM all-trans retinoic acid and collected at 0, 14 and 21 days. In parallel experiments, cells were replated at 14 days in Matrigel, deprived of retinoic acid and incubated with mitotic inhibitors and further cultured for 7 days. Control cells were maintained in initial retinoic acid treatment. Both PC12 and NT2N-collected cells were processed for total RNA extraction and evaluation of miRNAs expression levels using quantitative Real Time-PCR. A and B) miR-16, let-7a and miR-34a expression during PC12 and NT2N differentiation, respectively. miRNAs expression were evaluated from 10 ng of total RNA, using specific primers for each miRNA. GAPDH and RNU6B were used for normalization in PC12 and NT2N cells, respectively. Expression levels were calculated by the ΔΔCt method. NGF-untreated cells at 1 day was used as calibrator in PC12 cells, while undifferentiated (day 0) or unreplated cells were used as calibrator in NT2N cells. Data represent mean ± SEM of three independent experiments. A) *p < 0.05 and ^†p < 0.01 compared to 1 day. miR-16 and let-7a expressions in cells treated with 5 and 50 ng/ml NGF were also significantly different from NGF untreated cells at day 4 (p < 0.05) and day 2 (p < 0.01), respectively. B) *p < 0.05 and ^§p < 0.001 compared to day 0; ^†p < 0.05 and ^‡p < 0.001 compared to unreplated cells. d, days.