Fig. 7

Hsa-miR-301a-3p regulates GADD45A expression and mRNA stability during ER stress in an XBP1s-independent manner.XBP1s expression was induced in the HeLa S3 cell line [20] and XBP1s (A) and GADD45A (B) mRNA levels were monitored with qRT-PCR and normalized to RPLP0 mRNA levels and expressed as the fold change over control (no induction) samples. Data represents the mean ± SD of three independent experiments (3 replicates each). * P < 0.05 was considered significant. 16HBE14o- cells were transfected with pre-miR-301a and specific target protector- Tp (C, D and E) for the GADD45A binding site for hsa-miR-301a-3p or with anti-miR-301a-3p (F) or their respective controls and then treated with Tm (2.5 µg/ml) or Tg (50 nM) for 6 h. GADD45A mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA. Data represents the mean ± SD of three independent experiments (3 replicates each). * P < 0.05 was considered significant. G GADD45A mRNA half-life measurements were taken in 16HBE14o- exposed to Tm (2.5 µg/ml) in the presence or absence of 20 µM 4µ8C, as well as after pre-miR-301a transfection, and from the cells cultured under control conditions. Actinomycin D was added to stop transcription, after which the cells were collected, and total RNA was isolated and GADD45A mRNA levels at each time point were measured by real-time PCR and normalized to endogenous 18 S rRNA levels. RNA values for each time point were calculated from 2 individual samples generated in at least 2 independent experiments and measured in 4 technical replicates. Relative RNA levels at the time points indicated were plotted as differences from RNA levels at the initial time point (t = 0). The mRNA half-lives were calculated from the exponential decay using the trend line equation C/C₀ = e^–kdt (where C and C₀ are mRNA amounts at time t and at the t₀, respectively, and k_d is the mRNA decay constant). The error bars represent SD. The calculated parameters are provided in Supplemental Table 1