The results shown in this Chapter thus far indicate a relationship between miR-7, TGF-β1- dependent differentiation and E2 restoration of young fibroblast characteristics; all of which revolve around the EGFR-mediated branch of the differentiation pathway and the activation of the JAK/STAT1 signalling pathway. To further investigate this functional and mechanistic relationship, time-course experiments were performed and analysed by Western blotting and QPCR. TGF-β1 treatment over the course of 72 hours resulted in the gradual accumulation of phosphorylated STAT1, with no changes in the total STAT1 protein (Figure 5.7A). In contrast, phosphorylation of STAT1 decreased after 48 hours of E2 treatment; and this trend continued onto the 72 hour time-point (Figure 5.7B). Next, the mRNA expression of STAT1 was assessed. With TGF-β1 treatment, STAT1 mRNA was significantly upregulated at the 72 hour time point (Figure 5.7C), whilst E2 treatment had no significant affect on STAT1 mRNA expression (Figure 5.7D).
QPCR of EGFR mRNA and miR-7 under TGF-β1 and E2 treatments, assessed further consequences of changes in STAT1 activation. The levels of EGFR mRNA expression consistently decreased over the 72 hour time course, when cells were incubated with TGF-β1 (Figure 5.8A), the opposite was observed in cells incubated with E2 (Figure 5.8B). In contrast to EGFR expression, the expression of miR-7 was inversely correlated. miR-7 was consistently upregulated by TGF-β1, with a significantly increased 4-fold and 7-fold expression changes at 48 and 72 hours, respectively (Figure 5.8C). Additionally, miR-7 was significantly downregulated with E2, with a 2-fold and 4-fold decrease at 48 and 72 hours
respectively. (Figure 5.8D). It was therefore determined that 72 hours of treatment with E2 was sufficient to upregulate EGFR, whilst downregulating miR-7.
It was next investigated whether 72 hours of treatment with E2, followed by 72 hours of TGF-β1 was effective at restoring the differentiation response in aged fibroblasts. In cells that were treated with E2 followed by TGF-β1, expression of EGFR was maintained (Figure 5.9A) and expression of HAS2 significantly upregulated, compared to untreated controls (Figure 5.9B). The levels of miR-7 initially downregulated by E2 treatment, returned to those seen in untreated control cells, following TGF-β1 (Figure 5.9C). The myofibroblastic markers α- SMA (Figure 5.9D) and EDA-FN (Figure 5.9E), were both significantly upregulated when cells were incubated with E2, followed by TGF-β1. To test whether α-SMA stress-fibres were forming within the cells following treatments, the cells were permeabilised and stained for α- SMA (Figure 5.9F). In cells that had receieved both E2 and TGF-β1 treatments, there was a noticeable increase in the intensity of the α-SMA stain and although stress-fibres were not formed throughout the entire cell, there were bundles present. These data suggest that pre- treatment of cells with E2 can “prepare” aged cells for TGF-β1-dependent differentiation, through the upregulation of key components, EGFR and HAS2; and the down-regulation of the limiting factor, miR-7; indicating that optimisation of E2 treatments in combination with TGF-β1 could be beneficial for restoration of the differentiation response in cells nearing senescence.
Figure 5.1. Determination of the miR-7 Upregulation Pathway. Fibroblasts were grown to confluence,
growth arrested and incubated in serum-free media containing 0.6% (v/v) DMSO, 10µM SB431542 or 10µM AG1478 for 1 hour, before the media was replaced with serum-free media alone, media containing 10ng/ml TGF-β1, or media containing 10ng/ml EGF for 72 hours. RNA was extracted, purified, and reverse transcribed and then QPCR was used to analyse the mRNA expression of A. EGFR and B. α-SMA. C. miR- QPCR was used to analyse miR-7 expression. All results are shown as the mean ± s.e.m. of 3 independent experiments. **P=<0.01.
Figure 5.2. In Silico Analysis of the miR-7 Promoter Region. In silico analysis was used to determine A. the putative TSS for miR-7, dashed blue box indicates the
region predicted to contain the TSS and upstream promoter sequence. Image was generated and analysed using miRStart computational software (Institute of Bioinformatics and Systems, National Chiao Tung University, Hsinchu, Taiwan). B. Diagram of miR-7 coding region and upstream promoter region used for analysis, dashed blue box indicates region used for promoter studies. C. The promoter region for miR-7 was analysed for recognised transcription factor binding sites. Transcription factors of interest are labelled and underlined sections indicate the primers used for promoter amplification. Promoter sequence was analysed for transcription factors using MatInspector (Genomatix Software, Munich, Germany).
Figure 5.3. Analysis of miR-7 Promoter Activity. Fibroblasts were grown to 60-70% confluence and
transfected with either empty pGL3b (white bars) or miR-7-pGL3b (black bars). The indicated cellular treatments were performed 24 hours post-transfection and the promoter activity analysed after a further 24 hours. Co-transfection of Renilla was used to measure transfection efficiency and for normalisation of data. The results shown are the means of the firefly luciferase/Renilla luciferase ratios ± s.e.m. of 3 independent experiments. *P=<0.05, **P=<0.01.
Figure 5.4. Induction of STAT1 Activation and Expression. A. Young fibroblasts were grown to
confluence, growth arrested and stimulated with the indicated treatments for 72 hours, before STAT1 mRNA was assessed by QPCR. Results are shown as the mean ± s.e.m. of 3 independent experiments. B. Young or aged cells were grown to confluence and STAT1 mRNA was assessed by QPCR. Results are shown as the mean ± s.e.m. of 3 independent experiments. C. Young fibroblasts grown to confluence were growth arrested and incubated with the indicated treatments. Protein was extracted and Western blotting was used to assess the phosphorylation of STAT1. Total STAT1 protein was used as a loading control and for normalisation. The results shown are the mean ± s.e.m. of 3 independent experiments. *P=<0.05, **P=<0.01.
Figure 5.5. The Effects of Inhibiting the JAK/STAT Pathway. Fibroblasts were grown to confluence,
growth arrested and incubated with 15nM JAK Inhibitor I for 1 hour, prior to incubation in serum-free media alone, media containing 10ng/ml TGF-β1, or media containing 10ng/ml IFNγ for 72 hours. QPCR was used to analyse the expression of A. α-SMA mRNA, B. EGFR mRNA, C. HAS2 mRNA and D. miR-7. Results are shown as the mean ± s.e.m. of 3 independent experiments. *P=<0.05, **P=<0.01.
Figure 5.6. The Effects of E2 on Young and Aged Fibroblasts. Young or aged fibroblasts were grown to
confluence, growth arrested and incubated with serum-free media alone (white bars) or media containing 100nM E2 (black bars), for 72 hours. QPCR was used to analyse the expression of A. α-SMA mRNA, B. EGFR mRNA, C. HAS2 mRNA and D. miR-7. Results are shown as the mean ± s.e.m. of 3 independent experiments. E. Young (white boxes) and aged (black boxes) fibroblasts were incubated with E2 over 72 hours. At each indicated time-point, the proliferation rates were measured by the addition of 10% (v/v) alamarBlue for 1 hour, before fluorescent intensity analysis with an optical plate reader. *P=<0.05, **P=<0.01.
Figure 5.7. Regulation of STAT1 by TGF-β1 and E2. Fibroblasts were grown to confluence, growth arrested
and incubated with serum-free media alone, media containing 10ng/ml TGF-β1, or media containing 100nM E2 over 72 hours. Protein was extracted and Western blotting was used to assess the changes in phosphorylation of STAT1 with A. TGF-β1 treatment and B. E2 treatment. Total STAT1 protein was used to ensure equal loading and for normalisation of densitometry. Results shown are the mean ± s.e.m. of 3 independent experiments. QPCR was used to analyse the expression of STAT1 mRNA with C. TGF-β1 treatment and D. E2 treatment. Results are shown as the mean ± s.e.m. of 3 independent experiments. N/S= no
Figure 5.8. Regulation of EGFR and miR-7 by TGF-β1 and E2. Fibroblasts were grown to confluence;
growth arrested and incubated with serum-free media alone, media containing 10ng/ml TGF-β1, or media containing 100nM E2 over 72 hours. RNA was extracted and purified and then QPCR was used to analyse the expression of EGFR mRNA with A. TGF-β1 treatment and B. E2 treatment, and the expression of miR-7 with