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For simplicity, the fold changes are round off. For the complete number see Appendix B, Table 7.4, 7.5 and 7.6.

Fig. 4.4 The relative expression of the ten chosen miRNAs in the wt mice. Each miRNA have three bars

showing the fold change for each day: day 0, 3 and 10. The control was day 0 of the wt mice, which had a fold change equal to 1.

Fig. 4.4 shows the fold changes of the ten chosen miRNAs, in the wt mice. All of the fold changes from day 0 were used as controls, and were therefore equal to 1. Fig. 4.4 shows which miRNAs that were relatively increased or decreased compared to day 0, in the wt mice, of the different days. For instance, the relative expression of miR-126-3p was increased on both day 3, by a fold change of 1.52 and on day 10, by a 1.22 fold change, compared to day 0 (Fig. 4.4).

Page 32 of 69 The miRNAs that were increased on both day 3 and day 10 were miR-126-3p, miR-191, miR-320 and miR-342-3p by a fold change up to 1.5 on day 3 and a fold change up to 1.7 on day 10. miR- 133a, miR-193b and miR-150 were increased by a fold change up to 2.4 fold on day 3 but decreased by a fold change between 0.95 and 0.70 on day 10. The miRNAs that were decreased on day 3, but increased on day 10 were miR-1937c and miR-1937b by a fold change of 0.9 in both miRNAs on day 3 and by a maximum fold change of 1.27 fold on day 10. Finally, miR-574-3p was decreased on both day 3 and day 10 by a fold change of 0.88 and by a fold change of 0.4 respectively (Appendix B, Table 7.4) (Fig. 4.4).

Fig. 4.5 The relative expression of the ten chosen miRNAs in the wt STZ mice. Each miRNA has three bars

showing the fold changes for each day: day 0, 3 and 10. The control was day 0 of the wt STZ mice, which had a fold change equal to 1.

Fig. 4.5 shows the fold changes, for each of the ten miRNAs, of the wt STZ mice. The control was day 0 of the wt STZ mice. Fig. 4.5 shows the different fold changes of all the miRNAs, on day 3 and 10 compared to day 0. The miRNAs that were increased on both day 3 and day 10 were miR- 1937b and miR-1937c, by up to 24.8 folds on day 3 and up to 18.7 folds on day 10. miR-150 was increased on day 3 by 2.26 fold but decreased, by 2.2 folds, on day 10. The remaining miRNAs:

Page 33 of 69 miR-574-3p, miR-342-3p, miR-320, miR-193b, miR-191, miR-133a and miR-126-3p were

decreased, by 90 % and less, on both day 3 and day 10 (Appendix B, Table 7.5) (Fig. 4.5). By comparing the data from Fig. 4.4 and Fig. 4.5, it is shown that miR-1937b of the wt STZ mice was greatly increased on day 3 by a fold change of 5.28, but decreased on day 3 in the wt mice by a fold change of 0.94 on day 3 compared to day 0. This fold change was very close to 1, and was therefore not a notable change. Nevertheless, this fold change still signifies that the amount of miR- 1937b was decreased in comparison of the wt mice on day 0.

The relative expression of miR-1937c was in the two types of mice very different. In the wt STZ mice, miR-1937c had a fold change of 19.7 on day 10 and on day 3 it had a fold change of 25.76 (Fig. 4.5). Indicating a high increase in the expression of miR-1937c, in the wt STZ mice, on both day 3 and day 10 compared to day 0 of the wt STZ mice. Whereas, in the wt mice, there was a decrease by a fold change of 0.96 on day 3 and an increase by a 1.27 fold change on day 10 compared to day 0 of the wt mice (Fig. 4.4). This signifies that the change of the relatively

expression of miR-1937c was not notable on day 3 and had a very little change on day 10 in the wt mice.

It could be observed that, when comparing Fig. 4.4 and Fig. 4.5, the two different groups: the wt STZ mice and the wt mice were opposite, on day 3. The miRNAs were all except for miR-1937b, miR-1937c, miR-574-3p and miR-150, increased on day 3 of the wt mice, while they were

decreased in the wt STZ mice on day 3. miR-1937b and miR-1937c were increased in the wt STZ mice by a fold change up to 25.76 and decreased in the wt mice by a fold change less than 0.96 on day 3. miR-574-3p was decreased in both types of mice, by a fold change less than 0.9 and miR- 150 was increased in both types of mice by a fold change of 3.2 in the STZ mice and a fold change of 1.1 on day 3 (Appendix B, Table 7.4, 7.5).

However, these figures were not efficient in comparing the two different groups of mice. These data only indicated which miRNAs that were increased or decreased in the two separate groups of mice, compared to day 0. For comparing the two types of mice, they would need the same control. Therefore, new calculations of the fold changes were made, as mentioned earlier in this report. In these new calculations, day 0 the wt mice were used as control for every miRNA in both the wt STZ mice and the wt mice. Thus, the fold changes shows the relatively expression of miRNAs of both the wt STZ mice and wt mice, compared to day 0 of the wt mice.

Page 34 of 69 Fig. 4.6 The relative expression of the ten chosen miRNAs. Each miRNA has two bars showing the fold

changes for day 0, in the wt STZ mice compared to the the wt mice day 0. The control was day 0 of the wt mice, which had a fold change equal to 1.

Fig. 4.6 shows the fold changes of the ten chosen miRNAs on day 0, of the wt mice and the wt STZ mice. Day 0 of the wt mice were used as control. On day 0 of the wt STZ mice the miRNAs were all, except for miR-1937c and miR-1937b, increased by a fold change between 1.2 and 9.5, compared to the wt mice day 0. miR-1937c and miR-1937b, in the wt STZ mice, were the only miRNAs that were decreased on day 0, by a fold change of 0.04 and a fold change of 0.22, respectively (Fig. 4.6). 0 1 2 3 4 5 6 7 8 9 10 miR-126-3p miR-133a miR-191 miR-193b miR-320 miR-342-3p miR-574-3p miR-150 miR-1937c miR-1937b 2-∆∆Ct _{normalized to U6}

Fold changes of the ten miRNAs in wt mice & wt STZ

mice (control wt day 0)

Page 35 of 69 Fig. 4.7 The relative expression of the ten chosen miRNAs. Each miRNA has three bars showing the fold

changes for day 3, in the wt STZ mice and day 3 in the wt mice, compared to the wt mice day 0. The control was day 0 of the wt mice, which had a fold change equal to 1.

The miRNAs on day 3 did not have a general pattern. All of the fold changes had a big variation between the two groups: the wt STZ mice and the wt mice, unlike day 0 and day 10, where the miRNAs showed a more general pattern. Fig. 4.7 show the fold changes of ten chosen miRNAs on day 3 of the wt mice and the wt STZ mice, compared to the wt mice day 0.

The miRNAs that were increased, in both the wt STZ mice and the wt mice on day 3, were miR- 150, miR-126-3p, miR-133a and miR-191, by a fold change between 1.1 and 4.2. miR-1937c and miR-1937b was increased on day 3 in the wt STZ mice, by a fold change up to 2.5 and was both decreased, by a fold change of 0.9 in the wt mice on day 3. miR-193b, mir-320 and miR-342-3p were increased in the wt mice by maximum fold change of 1.3 and decreased by a change fold of 2.5 or less in the wt STZ mice. miR-574-3p was decreased by fold change of 0.88 in the wt mice and decreased by a fold change of 0.41 in the wt STZ mice, on day 3. (Appendix B, Table 7.5, 7.6) (Fig. 4.7).

Page 36 of 69 Fig. 4.8 The relative expression of the ten chosen miRNAs. Each miRNA has three bars showing the fold

changes for day 10, in the wt STZ mice and day 10 in the wt mice compared to day 0 in the wt mice. The control was day 0 of the wt mice, which had a fold change equal to 1.

Fig 4.8 show the ten miRNAs fold changes of the wt mice and the wt STZ mice on day 10,

compared to the wt mice day 0. There was a more general pattern on day 10. It showed, except for miR-191, that every miRNA was decreased among the wt STZ mice by up to 5 folds. In the wt mice, the fold changes varied more; miR-150, miR-574-3p, miR-193b and miR-133a were decreased by up to 3 folds. While the miRNAs miR-1937c, miR-342-3p, miR-320, miR-191 and miR-126-3p were increased, by up to 0.77 folds (Fig. 4.8).

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 miR-126-3p miR-133a miR-191 miR-193b miR-320 miR-342-3p miR-574-3p miR-150 miR-1937c miR-1937b 2-∆∆Ct _{normalized to U6}

Fold changes of the ten miRNAs in wt mice & wt STZ

mice (control wt day 0)

Page 37 of 69

5.1 THE TEN MIRNAS IN RELATION TO WOUND HEALING

Decrease or increase of several miRNAs influence wound healing by up- or down-regulating the expression of proteins that assist in wound repair or inhibit wound healing (Yi et al., 2008; Biswas et al., 2010; Chan et al., 2012b; Jin et al., 2013; van Solingen et al., 2014).

miR-320

Some of miR-320 targets’ are the proteins VEGF and fibronectin (Feng & Chakrabarti, 2012). The VEGF protein is secreted by macrophages in the inflammatory phase and is one of the growth factors that increase cell proliferation in the proliferative phase of wound healing. Furthermore the VEGF protein increases angiogenesis induction, vascular permeability, vasculogenesis, endothelial cell migration promotion and apoptosis inhibition (Genecards). Vasculogenesis is formation of new blood vessels derived from endothelial stem cells, when there are no pre-existing vessels (Maeda et al., 2008; Su et al., 2012). An up-regulation of miR-320 may results in decreased expression of the protein VEGF. This might indicate that cell proliferation in the proliferative phase is reduced, and that induction of angiogenesis and endothelial cell migration is impaired. New blood vessel formation might also be reduced when miR-320 is up-regulated. Apoptosis reduces the concentration of macrophages and fibroblast in the remodeling phase, so an up-regulation of miR- 320 might result in delayed macrophage and fibroblast reduction. The results of this experiment shows an up-regulation on day 3 and 10 of the wt mice compared to day 0, by a 1.3 and 1.8 fold change, respectively. The proliferative phase and the remodeling phase is after days to weeks, according to the theory VEGF is important on day 3 and 10 and therefore a down-regulation of miR-320 was expected. When comparing wt STZ mice with wt mice on day 0 there was a down- regulation by a 0.8 and 0.7 fold change on day 3 and day 10, respectively, this might indicate that more VEGF is expressed among wt STZ mice on day 3 and day10.

Fibronectin is also one of miR-320s’ targets and is important for host defense and blood coagulation in the hemostatic phase and for fibroblast migration and proliferation on the proliferative phase (Genecards). An up-regulation of miR-320 might result in reduced expression of fibronectin and thereby impaired blood coagulation, fibroblast migration and proliferation. As mentioned, the results of this project both showed an up-regulation on day 3 and 10 in wt mice