| Research BackgroundDue to trauma,tumor resection and other reasons,oral and maxillofacial skin wounds are very common in clinical practice.Similar to the skin traumas in other parts of the body,the management of oral and maxillofacial skin wound healing is still a big challenge.In the past,research on skin wound healing mainly focused on the epidermal cells.However,in recent years,more and more studies have shown that fibroblasts in the dermal tissue play a key role in wound healing process,especially in dermal remodeling and scarring formation.MicroRNAs(miRNAs)play important functions in different tissues and cells,and have found to be involved in all stages of wound healing.MicroRNA 21(MiR-21),one of the most widely studied microRNAs,has been reported to positively regulate the wound healing of mouse skin and periodontal tissues.However,the important role of miR-21 has been discovered in the skin wound healing,the role of miR-21 of dermal fibroblasts has not been studied.Moreover,previous studies for the role of miR-21 in skin wound healing have mainly focused on mouse models or mouse cells,but little is known the role miR-21 in human cells.Therefore,the present study firstly used miR-21 knock-out mice and corresponding wild-type littermates as control group to establish a skin wound model to observe and confirm whether the loss of miR-21 gene affects the healing of skin wound,especially the healing of dermal part of skin.Based on the in vivo study,human skin dermal fibroblast then was taken as the object of this study,and its miR-21 expression would be inhibited or promoted by transfection of small RNAs to investigate the role of miR-21 in the migration of human dermal and epidermal cells in vitro wound healing assay.And finally,the underlying molecular mechanism of miR-21 regulating skin cell migration and proliferation was further explored.Aims:(1)To verify whether the loss of miR-21 affects the wound healing of dermal tissues by using miR-21 knock-out mouse model in vivo.(2)To study the regulatory effect of miR-21 on the migration of dermal fibroblasts with inhibition or promotion of miR-21 expression through in vitro wound healing assay(scratch assay)and Transwell migration assay.(3)To perform RNA sequencing of dermal cells with miR-21 inhibition after scratching to explore its potential mechanism of regulating wound healing.(4)Further in vitro analysis to verify the role of dermal fibroblasts miR-21 in the regulation of skin wound healing via the identified molecular pathways by RNA sequencing.Methods:(1)The miR-21 knock-out and wildtype mice,were created 5mm diameter skin wounds by biopsy punch on the left and right sides of back skin after shaving.The wound healing of each mouse was monitored and taken photos every two days,and the size of non-healing was measured and analyzed by Image J software to calculate non-healing rate.(2)The healing skin after wounding was collected at different time points and processed for H&E staining and Masson staining to investigate the effect of miR-21 on the skin wound healing,especially the healing status of the dermal tissue.(3)The expression level of miR-21 in primary dermal fibroblasts derived from human skin was suppressed or promoted by transfecting corresponding small RNAs(miR-21 inhibitor and miR-21 mimics,unrelated RNA miR-21 NC as a control),and the effect of different expression levels of miR-21 on the migration of dermal fibroblasts were explored through in vitro scratch assay and Transwell assay as well,And at the same time the effect of miR-21 expression on the growth of dermal fibroblasts was studied by Cell Counting Kit 8(CCK-8).(4)After transfection of miR-21 inhibitor or RNA control(miR-21 NC),human dermal fibroblasts were scratched and the cells are collected at different times to extract RNA for RNA sequencing to identify the potential molecular pathways regulated by miR-21 to control dermal cell would closure in vitro scratch assay.(5)Based on the analysis results of above RNA sequencing,the conditioned medium of human dermal fibroblasts with inhibition or enhancement of miR-21 expression were collected and and used for the scratching or transwell experiments,and CCK-8 assay to verify that the potential secreted factors revealed by RNA sequencing which affect the proliferation and migration of skin cells,including dermal cells and epidermal cells,through a paracrine pathways.Results:(1)In vivo skin wound healing assay showed that the loss of miR-21 resulted in slow wound healing of skin,including both epidermal and dermal tissue,when compared with that of wild-type miR-21(+/+)control mice and the Masson staining showed that in the miR-21 knockout(-/-)mice,the collagen deposition was reduced and the arrangement of collagen was irregular after wound healing.These results suggest that the wound healing of the dermal tissue was significantly impaired in mice with loss of miR-21.(2)In vitro studies have shown that reducing the expression of miR-21 inhibits the migration of human dermal fibroblasts after scratching,while increasing the expression of miR-21 promotes the wound closure of human dermal fibroblasts after scratching.However,the expression level of miR-21 has no significant effect on the growth of dermal fibroblasts.(3)The analysis of RNA-seq results revealed that in vitro scratching assay of dermal fibroblasts,inhibition of miR-21 expression significantly inhibits the molecular pathway involving inflammatory response,associated with the lower expression of more than 60 cytokines,mainly including inflammatory cytokines.The selected 5 factors of them,LIF,LY 6E,IL-1β,IL-4R and TNFRSF1 B,were verified by quantitative RT-PCR.This result suggests that miR-21 may regulate the migration of dermal fibroblasts after wounding by regulating the expression of inflammatory factors.(4)The conditioned medium derived from scratched dermal fibroblasts with inhibition of miR-21 expression can inhibit the migration and proliferation of dermal and epidermal cells.On the contrary,the conditioned medium derived from cells with increased miR-21 expression can promote the migration and proliferation of dermal and epidermal cells.This result confirms that miR-21 in dermal cells can regulate the migration and growth of skin cells by a paracrine signaling pathway,possibly by regulating the expression and secretion of inflammatory cytokines.ConclusionThis study used the miR-21 knock-out mouse model to confirm that the loss of miR-21 in vivo delayed the healing of skin wounds,especially the healing of dermal tissue.In vitro studies have confirmed that the expression of miR-21 in dermal fibroblasts promotes the migration of dermal cells.Mechanically,we found that miR-21 in dermal fibroblasts could regulate the proliferation and migration of dermal fibroblasts and epidermal cells by regulating the secretion of related inflammatory factors(through paracrine signaling pathways).This study is the first time to explore the role of miR-21 in human dermal fibroblasts on skin cell wound healing.The research provides a solid foundation for the future study of miR-21 as a new target to promote skin wound healing and to screen of small molecule compounds targeting miR-21 to enhance dermal cell healing. |
PDF Full Text Request