| BackgroundWound healing is the process of restoring the structural integrity and physiological function of damaged skin,involving hemostasis,inflammation,reepithelialization and tissue remodeling.Among them,re-epithelialization is the process responsible for restoring the intact epidermis during the wound healing process,and this process is closely related to the proliferation and migration of human epidermal keratinocytes(HEKs).By promoting the proliferation and migration of HEKs cells,it can promote re-epithelialization,thereby promoting skin wound healing.Both circular RNA and microRNA are non-coding RNAs.Circular RNA plays the role of a molecular sponge that regulates the expression of the miRNA targets.Its target genes are associated with cell proliferation and differentiation,apoptosis,migration,and many other processes.The regulatory pathway of circRNA/miRNA/target genes plays an important role in human tumors,neurological diseases,cardiovascular and cerebrovascular diseases and other diseases,providing a new way for the elucidation of the pathogenesis of these diseases and the selection of therapeutic targets.Circular RNA DNA-dependent protein kinase(circ＿PRKDC)is a member of the circRNA family,and whether it can affect wound healing is unknown.Circular RNA Interactome target gene online software predicts that circ＿PRKDC may play the role of miR-31-5p molecular sponge;microT CDS target gene online software predicts that fibrillin-1(FBN1)may be the target gene of miR-31-5p.Whether circ＿PRKDC can target miR-31-5p/FBN1 axis to affect wound healing is still unknown.In order to explore the effect and the molecular mechanism of circ＿PRKDC on wound healing,this topic will mainly study from the following two parts:The first part is to observe the expression of circ＿PRKDC in the process of wound healing,and then the HEKs cells that overexpressed and knocked down circ＿PRKDC were constructed,we have observed The effect of expression or knockdown of circ＿PRKDC on the proliferation and migration of HEKs cells;the second part,taking the miR-31-5p/FBN1 axis as the starting point,explore the molecular mechanism of circ＿PRKDC affecting the proliferation and migration of HEKs.Part Ⅰ The expression of circ＿PRKDC during wound healing and its effect on the proliferation and migration of human epidermal keratinocytesObjectives:1.to clarify the differential expression of circ PRKDC during wound healing.2.to clarify the localisation of circ PRKDC in cells and its nature and structure.3.to clarify the effect of circ＿PRKDC on the proliferation and migration of human epidermal keratin-forming cells.Methods:1.Seven healthy volunteers(relative skin donors of patients with extensive burns)were selected for the study.During the first skin donor procedure,the thick skin was removed with a motorized knife and 3 mm x 3 mm skin tissue(mean thickness 0.3 mm)was left intact as a control skin(uninjured skin).Skin tissues were collected from the wounded edge of the donor area on day 1(inflammatory phase)and day 7(proliferative phase,at the time of the second skin donor)using the same method,and circ PRKDC expression was measured by qRT-PCR.2.HEKs cells were cultured in vitro,and RNase R experiment was use to observe whether circ＿PRKDC has a circular structure.After HEKs cells were treated with actinomycin D,the expression of circ＿PRKDC was detected by qRT-PCR method to observe the stability of circ＿PRKDC.observe circ＿PRKDC in HEKs by nucleoplasmic separation experiment Positioning in the cell.The localization of circ PRKDC in HEKs cells was observed by nuclear and cytoplasmic separation experiments.3.Empty vector(pCD5-ciR group),circ＿PRKDC overexpression vector(circ＿PRKDC group),small interfering RNA negative sequence(si-NC group),and circ PRKDC small interfering RNA(si-circ＿PRKDC group)were respectively transfected into HEKs cells,qRT-PCR method was used to detect the expression of circ＿PRKDC in HEKs cells to verify the transfection effect.Four groups of cells were inoculated into culture plates.In order to observe the proliferation of cells in each group,the number of EdU+cells and clones were detected by EdU experiment and clone formation experiment,respectively.And in order to observe cell migration in each group,the cell migration rate and migration number were detected by scratch test and Transwell chamber test.Western blotting was used to observe the protein expression levels of matrix metalloproteinase 2(MMP2)and matrix metalloproteinase 9(MMP9)in cells.Results:1.Compared with undamaged skin tissue,the expression of circ PRKDC was decreased during wound healing(P<0.05).1.circ＿PRKDC expression was reduced during wound healing compared to undamaged skin tissue,and the difference was statistically significant(P<0.05).2.circ＿PRKDC has a ring-like structure,is stable in nature and is mainly expressed in the cytoplasm of HEKs cells.3.In HEKs cells,the expression of circ PRKDC was increased in the circ＿PRKDC group compared with the pCD5-ciR group,and the difference was statistically significant(P<0.05).indicating that circ＿PRKDC was overexpressed in the circ＿PRKDC group in HEKs cells.4.The expression of circ＿PRKDC in the si-circ＿PRKDC group was reduced in HEKs cells compared with the si-NC group,and the difference was statistically significant(P<0.05).This indicates that circ＿PRKDC was knocked down in the sicirc＿PRKDC group in HEKs cells.5.The number of EdU+cells,the number of clones,the migration rate and migration number and the expression of MMP2 and MMP9 proteins in the cells in the circ＿PRKDC group were lower than those in the pCD5-ciR group,and the differences were statistically significant(P<0.05).6.The number of EdU+cells,the number of clones,the migration rate and migration number and the expression of MMP2 and MMP9 proteins in cells in the sicirc＿PRKDC group were all higher than those in the si-NC group,and the differences were statistically significant(P<0.05).Conclusions:1.circ PRKDC is circular,stable in nature,and is mainly expressed in the cytoplasm of human epidermal keratinocytes.2.The expression of circ＿PRKDC is down-regulated during wound healing.Overexpression of circ＿PRKDC inhibits the proliferation and migration of human epidermal keratinocytes,but knocking down circ＿PRKDC promotes the proliferation and migration of human epidermal keratinocytes.The second part circ＿PRKDC regulates the proliferation and migration of human epidermal keratinocytes by targeting the miR31-5p/FBN1 axisObjectives:1.To clarify the differential expression of miR-31-5p and FBN1 in the process of wound healing.2.To clarify the effect of circ PRKDC on HEKs cell proliferation and migration by targeting the miR-31-5p/FBN1 axis and the possible mechanism of the interaction.3.To clarify the effects and possible mechanisms of miR-31-5p and FBN1 on proliferation and migration of HEKs cells,respectively.Methods:1.Using circular RNA Interactome target gene online software prediction showed that circ＿PRKDC may competitively adsorb miR-31-5p;microT CDS target gene online software prediction showed that pro-fibronectin-1(FBN1)may be the target gene of miR-31-5p.The target regulation of circ＿PRKDC with miR-31-5p and miR-31-5p with FBN1 was verified using dual luciferase reporter gene assay,RNAbinding protein immunoprecipitation(RIP)assay and pull down assay.2.HEKs cells were divided into pCD5-ciR group,circ＿PRKDC group,si-NC group and si-circ＿PRKDC group.miR-31-5p expression was detected in each group by qRT-PCR,and the effect of circ＿PRKDC on miR-31-5p expression was observed.3.HEKs cells were divided into miR-NC group,miR-31-5p group,anti-miR-NC group and anti-miR-31-5p group.qRT-PCR and Western blotting assay were performed to detect the mRNA and protein expression of FBN1 in each group,and to observe the effect of miR-31-5p on the expression of FBN1.4.HEKs cells were divided into si-NC group,si-circ＿PRKDC group,sicirc＿PRKDC+pcDNA group and si-circ＿PRKDC+FBN1 group.qRT-PCR and Western blotting assays were performed to detect the mRNA and protein expression of FBN1 in each group of cells,respectively,to observe the effect of circ＿PRKDC on the expression of FBN1.5.qRT-PCR was used to detect the expression of miR-31-5p and FBN1 mRNA during wound healing,and Western blotting assay was used to detect FBN1 protein expression.Spearman correlation analysis of circ＿PRKDC and miR-31-5p,miR-315p and FBN1 mRNA and circ＿PRKDC correlated with FBN1 mRNA expression.6.HEKs cells were divided into si-NC group,si-circ＿PRKDC group,sicirc＿PRKDC+anti-miR-NC group and si-circ＿PRKDC+anti-miR-31-5p group,and the cell EdU+cell number and clone number were detected by EdU assay and clone formation assay,respectively,and the scratch assay and Transwell assay to detect cell migration rate and migration number,respectively,and Western blotting to detect MMP2 and MMP9 protein expression.7.HEKs cells were divided into miR-NC group,miR-31-5p group,miR-315p+pcDNA group and miR-31-5p+FBN1 group,and the number of EdU+cells and clones were detected by EdU assay and clone formation assay,and the cell migration rate and migration number were detected by scratch assay and Transwell assay,respectively.Western blotting assay was used to detect MMP2 and MMP9 protein expression.8.HEKs cells were divided into si-NC group,si-circ＿PRKDC group,sicirc＿PRKDC+pcDNA group and si-circ＿PRKDC+FBN1 group,and the number of EdU+cells and clones were detected by EdU assay and clone formation assay,and the cell migration rate and migration number were detected by scratch assay and Transwell assay,respectively.MMP2 and MMP9 protein expression were detected by Western blotting assay.Results:1.circ＿PRKDC can target miR-31-5p,and FBN1 is the target gene of miR-315p.2.The expression of miR-31-5p in HEKs cells in the circ PRKDC group was lower than that in the pCD5-ciR group(P<0.05),but the expression of miR-31-5p in HEKs cells in the si-circ＿PRKDC group was higher than that in the si-NC group(P<0.05).The expression of FBN1 mRNA and protein in HEKs cells in the miR-315p group were lower than those in the miR-NC group(P<0.05),but the expression of FBN1 mRNA and protein in HEKs cells in the anti-miR-31-5p group were higher than those in the anti-miR-31-5p group.The expressions of FBN1 mRNA and protein in HEKs cells in the si-circ＿PRKDC group were significantly lower than those in the si-NC group(P<0.05),but the expression of FBN1 mRNA and protein in HEKs cells in the si-circ＿PRKDC+FBN1 group were significantly higher than those in sicirc＿PRKDC+pcDNA group(P<0.05).3.Compared with undamaged skin tissue,the expression of miR-31-5p was increased during wound healing(P<0.05),but the expression of FBN1 mRNA and protein were decreased during wound healing(P<0.05).The results of Spearman correlation analysis showed that the expression of circ＿PRKDC in damaged skin tissue was negatively correlated with the expression of miR-31-5p(r=-0.911,P<0.05),and positively correlated with the expression of FBN1 mRNA(r=0.787,P<0.05).The expression of miR-31-5p in damaged skin tissue was negatively correlated with the expression of FBN1 mRNA(r=-0.790,P<0.05).4.The EdU+cell number,clone number,migration rate and migration number in the si-circ＿PRKDC group and the protein expression of MMP2 and MMP9 were higher than those in the si-NC group(P<0.05),but the EdU+cell number,clone number,migration rate and migration number in the si-circ＿PRKDC+anti-miR-31-5p group and the protein expression of MMP2 and MMP9 were lower than those in the si-circ＿PRKDC+anti-miR-NC group(P<0.05).5.The EdU+ cell number,clone number,migration rate and migration number in the miR-31-5p group and the protein expression of MMP2 and MMP9 were all higher than those in the miR-NC group(P<0.05),but the EdU+cell number,clone number,migration rate and migration number in the miR-31-5p+FBN1 group and the protein expression of MMP2 and MMP9 were were all lower than those in the miR-315p+pcDNA group(P<0.05).6.The EdU+ cell number,clone number,migration rate and migration number in the si-circ＿PRKDC group and the protein expression of MMP2 and MMP9 were higher than those in the si-NC group(P<0.05),but the EdU+cell number,clone number,migration rate and migration number in the si-circ＿PRKDC+FBN1 group and the protein expression of MMP2 and MMP9 were all lower than those in the sicirc＿PRKDC+pcDNA group(P<0.05).Conclusions:1.The expression of miR-31-5p increases during wound healing,but the expression of FBN1 decreases.In damaged skin tissue,the expression of circ＿PRKDC is negatively correlated with miR-31-5p,while mRNA expression of FBN1 is negatively correlated with miR-31-5p,but the expression of circ＿PRKDC was positively correlated with the expression of FBN1 mRNA.2.MiR-31-5p targets and negatively regulates FBN1,circ＿PRKDC targets and negatively regulates miR-31-5p,and circ＿PRKDC positively regulates FBN1.3.Down-regulating miR-31-5p reverses the effect of knocking down circ＿PRKDC on the proliferation and migration of HEKs cells.4.Proliferation and migration of HEKs cells is facilitated by upregulation of miR-31-5p,but overexpression of FBN1 reverses the effect of overexpressing miR31-5p on the proliferation and migration of HEKs cells.5.Overexpression of FBN1 reverses the effect of knocking down circ＿PRKDC on the proliferation and migration of HEKs cells.Full text conclusion:1.The expression of circ＿PRKDC and FBN1 are decreased during wound healing,but the expression of miR-31-5p is increased.2.Knockdown of circ PRKDC can promote the proliferation and migration of human epidermal keratinocytes by targeting miR-31-5p and then down-regulating the expression of FBN1.circ＿PRKDC may become a molecular target to promote wound healing. |
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