| Objective:High-throughput miRNA array technology was used to screen miRNAs differentially expressed in denatured dermis compared with normal skin, then predicted their target genes and the possible signaling pathways involving of wound healing by bioinformatics methods. We further detected the expression of miR-199a-5p and miR-126in rat denatured dermis. Next, we studied the role of miR-199a-5p in thermal damaged fibroblasts recovery and the role of miR-126in thermal damaged HUVECs recovery, and explored the possible mechanisms.Method:1. Collection of denatured dermis in burn patients and the matched normal skin, and all the specimens were collected at the fourth day after burns. High-throughput miRNA microarray technology was used to test the differences expressed miRNAs between denatured dermis and normal skin. To verify the chip results, some differentially expressed miRNAs were examined by QRT-PCR technology again. Then predicted their target genes and the possible signaling pathways involving of wound healing by bioinformatics methods.2. Using QRT-PCR to detect the expression of miR-199a-5p and miR-126at different times in the rat denatured dermis (1day,3days,5day,7day,14day after burns)3. Establishion of fibroblasts thermal damage model, then application of QRT-PCR to detect the expression of miR-199a-5p in thermal damaged fibroblasts at different times (2h、24h、48h). After transfected with miR-199a-5p mimics and inhibitors, fibroblasts were suffered to thermal damage, then to observe the effects of miR-199a-5p on the proliferation and migration of thermal damaged fibroblasts by MTT assay, cell scratch test and Transwell assay.4. Establishion of HUVECs thermal damage model, then application of QRT-PCR to detect the expression of miR-126in thermal damaged HUVECs at different times (2h、12h、24h). After transfected with miR-126mimics and inhibitors, HUVECs were suffered to thermal damage, then to observe the effects of miR-126on the proliferation and migration of thermal damaged HUVECs by MTT assay, cell scratch test and Transwell assay.Result:1. A total of66miRNAs were differentially expressed in denatured dermis compared with normal skin,among which34were down-regulated while32are up-regulated. The most significantly up-regulated miRNA was miR-223, and the most significantly down-regulated was miR-203. Differentially expressed miRNAs were predicted to be related with several signalling pathways in wound healing.2. By making rat deep second degree burns model, we found that the expression of miR-199a-5p was decreased in the rat denatured dermis at early stage, but it returned to normal at the seventh day; the expression of miR-126in the rat denatured dermis was significantly lowered, and returned to normal at5th day after injury, but increased to5times of the normal skin at the14th day.3. After transfected with miR-199a-5p mimics or inhibitors, fibroblasts were suffered to thermal damage. The results showed that: fibroblasts in group of miRNA-199a-5p inhibitor grew significantly faster by MTT assay, moved more distance by cell scratch test, migrated larger number of cells by transwell experiment compared with the control group (P<0.05). All the experimental data, there was no significant difference between the group of miRNA-199a-5p mimic and control group(P>0.05).4. HUVECs were transfected with miR-126mimics or inhibitors, then suffered the pretreatment of thermal damage. MTT results showed that:cells in group of miRNA-126mimics grew significantly faster than the control group (P<0.05), especially in the24hour and48hours performance significantly; the cell scratch experiments showed:cells in the group of miRNA-126mimics moved further distance; the transwell experiment results showed that:miRNA-126mimics group migrated larger number of cells (P<0.05). There was no significant difference between miRNA-199a-5p inhibitor group and control group in experimental data.(P>0.05).Conclusion:1. Identified differentially expressed miRNAs between denatured dermis of the burn patients and normal skin, and established a foundation for further studying the mechanism of denatured dermis recovery.2. The expression of miR-199a-5p and miR-126in rat denatured dermis showed a time-dependent changes. The expression of both miR-199a-5p and miR-126were decreased at early stage, then gradually returned to normal, the difference was that the expression of miR-126was going on increased significantly than that normal skin at late stage.3. At the early stage of fibroblasts thermal damage, the expression of miR-199a-5p was declined, then return to normal later. The low expression of miRNA-199a-5p could promote the proliferation and migration of thermal damaged fibroblasts.4. At the early stage of HUVECs thermal damage, the expression of miR-126was declined, then return to normal later, and going on increased significantly. The high expression of miR-126could enhance the proliferation and migration of thermal damaged HUVECs. |
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