| Skin is the first barrier to protect human body from external stimulation and injury.Skin injury caused by various acute and chronic injury factors is very common in clinic.Skin wound healing is a complex dynamic process,which involves three overlapping stages:inflammation,proliferation and remodeling.Severe wound healing often leads to excessive deposition and excessive fibrosis of collagen,and scar formation,which seriously affects patient's mental health and physiological function,so wound healing caused by various kinds of wounds is still a serious medical and social problem.In recent years,a class of small non-coding RNA--microRNA,which regulate protein expression at post-transcriptional level,has been found to be involved in many biological functions,and their role in wound healing has also been gradually discovered and valued.A large number of studies have found that there are many differentially expressed miRNAs in the process of skin wound healing,and some miRNAs are involved in the three complex dynamic repair processes:inflammation,proliferation and remodeling in wound healing.It has been reported that miR-203 and mir-210could induce wound healing by mediating the proliferation and migration of keratinocytes,while miR-99 and miR-200 families inhibited wound healing by depressing keratinocyte migration.Other studies have also found that cancer-related miRNA——miR-21,which is closely related to tumorigenesis and progression,is significantly up-regulated in skin wounds.Inhibition of miR-21 expression can significantly inhibit wound healing and collagen deposition,whereas overexpression of miR-21 can promote wound healing.At the same time,a large number of studies have confirmed that miR-21 can promote tissue fibrosis in lung,kidney and liver,and is also highly expressed in hypertrophic skin scar,which indicated that miR-21may serve as a stress protective molecule to promote wound healing after wound injury.In addition,it has been proved that the differentiation and function of Dendritic cells?DCs?in peripheral blood can also be regulated by miR-21.Interestingly,recent research report that DCs play an important role in wound repair,incresing the content of DCs significantly promoted wound healing,while reducing the content of DCs showed delayed wound healing.Therefore,we speculated that if overexpression of miR-21 in wound tissue can promote wound healing by increasing the content of DCs in wound tissue?To explore the role of miR-21 in skin wound healing and its potential molecular mechanism,lentivirus bearing miR-21 mimic or inhibitor were injected into the peripheral tissue around wounds to modulate the expression of miR-21 in wound tissues.Take these rats as the research object,to explore the effects of miR-21 on the content of DCs in wound tissue,wound healing,ralated index and signal pathway PTEN/PI3K/AKT were observed in vivo;the effects of miR-21 on DCs differentiation and keratinocyte migration were further studied by in vitro cell model.Part I Expression of miR-21 and related indexes in rat wound tissueMethodsTwenty-five clean male SD rats were fed in room for 7 days,then anesthetized by intraperitoneal injection of 1%pentobarbital sodium?350 mg/kg?,and fixed on the operating table.Hair removal was carried out on the 7 cm×3 cm area of the back,and iodophor alcohol was disinfected.A 4 cm2 full-thickness skin defect wound was made with an aseptic scalpel,and the skin was cut completely,the wound was opened,reached to the muscles and fully stopped.Then the wound was covered with aseptic gauze,and fixed with external medical tape.All SD rats eat and drink freely.Five SD rats were randomly selected at 6h,24h,48h and 72h after wounds model establishment.The wound fluids were collected from each rat to detect the cell content,and the wound tissues were taken for molecular biological detection.ResultsMiR-21 was upregulated at 0,6,24,48,72 h after model establishment,and the increase reached a maximum at 24 h in wound tissues;MMP-9 expression presented the same tread as miR-21 and was significantly enhanced within 6 h of wound formation,and then remain increased to the maximum at 24 h.The cell total number in wound fluids also markedly increased within 72 h of wound formation,while accompanied by the increase of percentage of CD11c+CD209+DCs.Further analysis indicated that miR-21 was positively correlated to MMP-9 expression?r=0.9027?,as well as the percentage of CD11c+CD209+DCs?r=0.9348?.The expression of PTEN protein was decreased within 72 h of wound formation;while p-AKT protein expression was markedly increased.ConclusionsAfter wounds establishment,the expression of miR-21,MMP-9,p-AKT,and the total number of cells and the content of CD11c+CD209+DCs in wound fluids were increased significantly and reached the maximum at 24 h,while the expression of PTEN in wound tissue decreased significantly and reached the minimum value at24h.In addition,the expression of miR-21 was positively correlated with the expression of MMP-9 and the content of CD11c+CD209+DCs.Part II MiR-21 accelerated skin wound healingMethodsLentivirus vectors?LV-miR-21 mimic and LV-miR-21 inhibitor?and packaging plasmids were transfected into 293T cells to package lentivirus.One hundred and sixty male Sprague–Dawley?SD?rats?12 weeks old,weighing 220–250 g?of clean grade after establishing wounds were randomly divided into 4 groups:control group,LV-NC group,LV-miR-21 mimic group and LV-miR-21 inhibitor group,40 rats in each group.And then,5?l physiological saline solution was injected into the peripheral tissue around wounds of control group SD rats.The other groups were injected with 5?l LV-NC lentivirus,LV-miR-21 mimic lentivirus or LV-miR-21inhibitor lentivirus,respectively,to modulate the expression of miR-21 in wound tissues.The wound surface of rats was covered with transparent film after wounds establishment.The area of the wound was statistically analyzed by image analysis software image-proplus6.0 after taking pictures by digital camera.The wound residual rate was measured daily for 15 days beginning from before injection.The average at each wound site of five rats was calculated and determined to be the average residual rate for that wound:the wound residual rate=Ss/Si×100%,where Si represents the initial area and Ss was the area of the wound at a specified time.The changes of miR-21 and PTEN mRNA in wound tissues were detected by qRT-PCR,the content of CD11c+CD209+DCs in wound fluids was detected by flow cytometry.Western blot was used to detect the protein level of PTEN,p-AKT,AKT and MMP-9 in wound tissue.ResultsCompared with the control group,the expression of miR-21 in wound tissues was obviously up-regulated by infection with LV-miR-21 mimic,but decreased with the infection of LV-miR-21 inhibitor.Compared with the control group,LV-miR-21mimic significantly improved the healing of skin wounds in rats;while LV-miR-21inhibitor delayed wound healing process.Furthermore,the percentage of CD11c+CD209+DCs increased with the increased of miR-21 but decreased with the decreased of miR-21.However,LV-miR-21 mimic significantly inhibited PTEN expression,while PTEN was promoted by LV-miR-21 inhibitor both in mRNA and protein levels.MiR-21 overexpression also elevated p-AKT and MMP-9 expression at protein level;by contrast,p-AKT and MMP-9 expression were attenuated by miR-21 knockdown.ConclusionsOverexpression of miR-21 could obviously shorten the time of skin wounds healing in rats,induce the enrichment of CD11c+CD209+DCs and the expression of p-AKT,MMP-9 in wound tissues,while inhibit the expression of PTEN in wound tissues.On the contrary,inhibiting miR-21 expressiom could significantly delay the process of wound healing,reduce the content of CD11c+CD209+DCs,the expression of p-AKT and MMP-9 in wound tissues,while the expression of PTEN in wound tissue was increased.In summary,these results confirmed that miR-21could promote skin wound healing of rats,otherwise,the wound healing will be delayed.Part III MiR-21 regulated wound healing via increasing DCs MethodsLentivirus vectors?LV-miR-21 mimic and LV-miR-21 inhibitor?and packaging plasmids were transfected into 293T cells to package lentivirus.16 male Sprague–Dawley?SD?rats?12 weeks old,weighing 220–250 g?of clean grade after establishing wounds were randomly divided into 4 groups:control group,FL group,LV-miR-21 inhibitor group and LV-miR-21 inhibitor+FL group,15 rats in each group.And then,5?l physiological saline solution was injected into the peripheral tissue around wounds of control group SD rats.LV-miR-21 inhibitor group and LV-miR-21 inhibitor+FL group were injected with 5?l LV-miR-21 inhibitor lentivirus,to modulate the expression of miR-21 in wound tissues.The FL group and LV-miR-21 inhibitor+FL group were injected with FMS like tyrosine kinase-3 ligand?FL?into the peripheral dermis tissue around the wound tissues to stimulate the differentiation of DCs,10?g per day for 4 consecutive days.The surface of wound was covered with transparent film after wounds establishment.The area of the wound was statistically analyzed by image analysis software image-proplus6.0 after taking pictures by digital camera.The wound residual rate was measured at 1,3,5 day beginning from before injection.The average at each wound site of five rats was calculated and determined to be the average residual rate for that wound:the wound residual rate=Ss/Si×100%,where Si represents the initial area and Ss was the area of the wound at a specified time.The changes of miR-21 and PTEN mRNA in wound tissues were detected by qRT-PCR,the content of CD11c+CD209+DCs in wound fluids was detected by flow cytometry.Western blot was used to detect the protein level of PTEN,p-AKT,AKT and MMP-9 in wound tissue.ResultsContrary to LV-miR-21 inhibitor,FL could significantly promote the healing of skin wounds,and eliminated LV-miR-21 inhibitor blockade of wound healing?Fig.3A?.FL facilitated the increase of CD11c+CD209+DCs ratio and promoted miR-21,p-AKT,and MMP-9 expression,but inhibited PTEN.Moreover,FL was able to reverse the effect of LV-miR-21 inhibitor on CD11c+CD209+DCs ratio,miR-21,p-AKT,MMP-9 and PTEN expression.ConclusionsFL could induce the increase of CD11c+CD209+DCS content in wound tissue,promote the expression of miR-21,p-Akt and MMP-9,inhibit the expression of PTEN and reverse the effect of LV-miR-21 inhibitor,indicating that miR-21 can promote the healing of the skin wounds by increasing the content of DCs in the wound tissue.Part IV MiR-21 promoted DCs differentiationMethodsPeripheral blood mononuclear cells?PBMCs?were isolated from rat peripheral blood by using lymphocyte isolate.The isolated PBMCs was cultured in the RPMI1640 medium containing 10%FBS and 1%streptomycin penicillin in a humidified heat preservation cell incubator with 37°C,5%CO2.PBMCs were transfected with miR-21 mimics/inhibitors or miR-NC,and then cultured with recombinant rat GM-CSF and IL-4 for 5 days.The content of CD11c+CD209+DCs in PBMCs was detected by flow cytometry,the changes of miR-21,PTEN and MMP9 mRNA were detected by qRT-PCR.Western blot was used to detect the protein level of PTEN,p-AKT,AKT and MMP-9 in PBMCs.The secretion of MMP-9 in PBMCs was detected by ELISA.ResultsMiR-21 silencing inhibited the differentiation of DCs in PBMCs,while miR-21overexpression obviously promoted DCs differentiation.MiR-21 inh ibitor evidently up-regulated the expression of PTEN,while PTEN was inhibited by miR-21overexpression both in mRNA and protein levels.MiR-21 inhibitor decreased the secretion of MMP-9 and p-AKT;while miR-21 mimic had the opposite effect on MMP-9 and p-AKT.ConclusionsOverexpression of miR-21 could promote the differentiation of DCs in PBMCs,inhibit the expression of PTEN,promote the expression of p-AKT and MMP-9,and induce the increase of MMP-9 secretion in the supernatant.Part V PTEN regulated DCs differentiation through PI3K/AKT MethodsPeripheral blood mononuclear cells?PBMCs?were isolated from rat peripheral blood by using lymphocyte isolate.The isolated PBMCs was cultured in the RPMI1640 medium containing 10%FBS and 1%streptomycin penicillin in a humidified heat preservation cell incubator with 37°C,5%CO2.PBMCs were transfected with si-PTEN and treated with or without PI3K/AKT inhibitor LY294002,and then cultured with recombinant rat GM-CSF and IL-4 for 5 days.The content of CD11c+CD209+DCs in PBMCs was detected by flow cytometry,the secretion of MMP-9 in PBMCs was detected by ELISA.Western blot was used to detect the protein level of p-AKT and AKT in PBMCs.ResultsPTEN knockdown significantly improved the differentiation of DCs and secretion of MMP-9 in the culture medium,as well as p-AKT/AKT,but PI3K/AKT inhibitor LY294002 markedly alleviated the increase of DCs ratio,MMP-9 secretion,and p-AKT/AKT induced by si-PTEN.ConclusionsPTEN could inhibit the differentiation of DCs in PBMCs and the secretion of MMP-9 in supernatant through PI3K/AKT signaling pathway.Part VI MiR-21 promotes DCs differentiation by regulating PTEN and thus affects the migration of keratinocytesMethodsTargetscan online bioinformatics analysis website and dual luciferase reporter gene experiment were used to verify the targeted regulatory relationship between miR-21 and PTEN.Peripheral blood mononuclear cells?PBMCs?were isolated from rat peripheral blood by using lymphocyte isolate.The isolated PBMCs was cultured in the RPMI 1640 medium containing 10%FBS and 1%streptomycin penicillin in a humidified heat preservation cell incubator with 37°C,5%CO2.PBMCs were transfected with miR-21 inhibitors and/or si-PTEN,and then cultured with recombinant rat GM-CSF and IL-4 for 5 days.The secretion of MMP-9 in PBMCs was detected by ELISA.Using supernatants from transfected PBMCs to culture keratinocytes,and wound healing assay to detect the migration of keratinocytes.ResultsBioinformatics analysis showed the complementary base pairs between miR-21and PTEN;luciferase activity of PTEN was inhibited by miR-21 mimic in analysis of luciferase reporter gene.The supernatant culture medium of the miR-21 inhibitor transfected cells markedly inhibited the migration ability of keratinocyted,but si-PTEN significantly reversed the role of miR-21 silencing in the migration ability of keratinocytes.Futher studies showed that si-PTEN reduced the relative expression of MMP-9;whereas the down-regulation could be apparently attrnuated by si-PTEN.ConclusionsMiR-21 could promote the differentiation of DCs by targeting the expression of PTEN and thus affect the migration of keratinocytes.SummaryIn conclusion,we demonstrated that the protective effect of miR-21 on wound healing was mediated,at least in part,via inhibition of PTEN which activated AKT/PI3K signaling pathway to increase DCs.MiR-21 treatment has a profound impact on wound healing.The present study may provide a basis for novel therapeutic strategies aimed at DCs enhancement in wound healing. |
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