The Roles Of MiR-198, MiR-203, MiR-210 And MiR-34a In Cutaneous Wound Healing

BackgroundSkin wound healing is a complex process involving the differentiation and proliferation of epidermal stem cells, and typical skin wound healing begins with the migration of keratinocytes on the edge of wound, while the re-epithelialization also involves the follicular cells there. Generally, wound healing recapitulates in certain extent the process of development and shares common cell biological and molecular features with cancer cells. Mi RNA is an important regulator in skin. MiR-198 is recently reported as a tumor suppressor by repression of mitogenic and motogenic pathways diminishing cell growth, while the potential effect of mi R-198 on cell proliferation is poorly understood in non-cancer tissues or cells. MiR-203 is an epitheliar tissue specific miRNA, which not only participates in the development of skin, but also regulates cell behavior as a tumor suppressor and apoptosis promoter. MiR-210 is a hypoxia-related mi RNA that existed in kinds of tissues and cells, and its expression plays key role in cell proliferation, mitochondrial respiration, DNA repair, and angiogenesis. MiR-34 a is a cancer-related mi RNA with plenty of researches.The prevention and treatment of skin exposure to sulfur mustard is constantly the emphasis of vesicant research. Keratinocytes, especially basal cells, are the main target cells of sulfur mustard. The mechanism of sulfur mustard injury is not yet clearly understood, and there is no research on the relationship between mi RNA and sulfur mustard yet.ContentAim 1. To evaluate the expression changes of miR-198 when the HaCaT cells, a cell line of immortalized human keratinocytes, exposed to sulfur mustard, to assess the effect of miR-198 overexpression on HaCaT cell proliferation and cell cycle progression, to search a new potential target of mi R-198 by bioinformatics prediction tools and identify the target by luciferase assay,to test whether this new target would regulate cell proliferation and cell cycle. Moreover, to evaluate the effect of mi R-198 and its new target on cell migration as well as adhesion. Finally, to predict and identify mmu-mi R-198 using bioinformatics and experimental tools.Aim 2. To detect the expression of mi R-203 in HaCaT cells and SKH-1 mouse skin after the exposure of sulfur mustard, to assess the effect of mi R-203 on cell proliferation and migration as well as their survival capacity after sulfur mustard injury in cultured cells. To explore the role of miR-203 expression changes in mouse skin wound healing of excision injury and sulfur mustard injury, and its potential protective effect on bone marrow and small intestinal epithlium in animal model, to identify the effect of miR-203 promoter methylation on cancer development by collecting and analyzing clinical data, and explore the possibility of miR-203 as a biomarker in cancer diagnosis and prognosis.Aim 3. To detect the expression of mi R-210 in Ha CaT cells and SKH-1 mouse skin after the exposure of sulfur mustard, to assess the effect of mi R-210 on cell proliferation and migration in in vitro assay, to explore the role of mi R-210 expression changes in mouse skin wound healings of excision injury and sulfur mustard injury, and its potential protection effect on bone marrow and small intestinal epithlium, to explore the possibility of miR-210 as a biomarker in cancer prognosis by collecting and analyzing clinical data.Aim 4. To explore the possibility of miR-34 a as a biomarker in cancer prognosis by collecting and analyzing clinical data.Result1. The expression of mi R-198 was significantly elevated in HaCaT cells after exposed to sulfur mustard. Transient transfection of mi R-198 mimics leads to HaCa T cell proliferation inhibition and cell cycle arrest. Bioinformatic al predictions identified cyclin D2(CCND2) as a new potential target of miR-198, and luciferase assay results showed that miR-198 could reduce CCND2 expression at both m RNA and protein level by directly binding to its 3‘-Untranslated Regions(3‘-UTR) as indicated by the site mutation experiment. The reduced CCND2 in cells inhibited cell proliferation and arrested cell cycle, which resemble the overexpresion of miR-198. However, miR-198 promotes cell migration and adhesion, while CCND2 repression did not show a similar effect. The prediction and identification of mmu-mi R-198 was tried in vain.2. Mi R-203 expression did not showed significant change in Ha CaT cells exposed to sulfur mustard, while its expression changed a lot in sulfur mustard treated mouse skins. In cells, mi R-203 repressed cell proliferation and migration, and which may participate the wound healing process in mouse skin maturation. Forced expression of mi R-203 did not show promotion to skin wound healing, but the reduced expression of mi R-203 delayed the healing process. Both mi R-203 overexpression and repression showed similar protective effect on marrow and intestine of mouse. Clinical data showed that mi R-203 methylation was responsible to the abnormal mi R-203 expression in cancer patients, while based on the current results, mi R-203 was not an ideal biomarker for cancer diagnosis and prognosis.3. Mi R-210 expression showed significant elevation in HaCaT cells exposed to sulfur mustard, its overexpression promoted cell proliferation and migration, and the reduced expression of miR-210 showed the opposite results. In mouse skin, increased miR-210 expression was also detected. Reduced expression of mi R-210 showed significant repression to the healing in early and middle period and tiny protective effect on marrow and intestine of mouse. Clinical data showed the abnormal miR-210 expression in cancer patients, while the elevated expression of mi R-210 predicted poor survival of cancer patients.4. Mi R-34 a showed a significant expression changes in various cancers. Most researchers identified the predictive effect of overexpression of mi R-34 a on good prognosis of cancer patients, while there were also researches showed the opposite results. After meta-analysis, we believed that miR-34 a over expression could predict good survival in several cancers.ConclusionMiR-198, mi R-210 expressions were elevated when Ha CaT cells was exposed to sulfur mustard, while the expression of miR-203 showed insignificant change. MiR-198 inhibited cell proliferation and led to cell cycle arrest by directly targeting CCND2. MiR-203 expression promoted cell survival in sulfur mustard treated HaCaT cells, and its effect on cell migration was also obvious. MiR-210 repressed HaCaT cell proliferation and migration. In the sulfur mustard exposed mouse skin, mi R-203 and mi R-210 expression dramatically exchanged, and reduced mi R-203 expression inhibited skin wound healing, while the reduced expression of mi R-210 promoted the healing process. Clinical data analysis showed that, mi R-210 and miR-34 a were both relative ideal biomarker for cancer patients prognosis, while miR-203 was not.